Patent Publication Number: US-2020277659-A1

Title: Novel processes for the production of oligonucleotides

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     This application claims the benefit of U.K. Provisional Application No. GB 1612011.5, filed 11 Jul. 2016. 
     FIELD OF THE INVENTION 
     The invention relates to novel processes for the production of oligonucleotides that are suitable for use in the production of chemically modified oligonucleotides, such as those used in therapy. 
     BACKGROUND TO THE INVENTION 
     The chemical synthesis of oligonucleotides and modified oligonucleotides via phosphoramidite chemistry is well established and has been the method of choice for synthesizing these defined sequence biopolymers for several decades. The synthetic process is usually run as a solid phase synthesis whereby single nucleotides are added sequentially with the addition of each nucleotide requiring a cycle of several chemical steps to add and deprotect the growing oligonucleotide (“oligo”) in preparation for the subsequent step. At the end of the sequential addition of nucleotides the oligo is released from the solid phase support, further deprotection takes place, and then the crude oligonucleotide is further purified by column chromatography. 
     While this method may be considered routine and can be automated, there are several shortcomings to this methodology, especially if the goal is to prepare oligonucleotides at large scale as would be needed for oligonucleotide therapeutics. These shortcomings include, but are not limited to:
         1) Practical limitations inherent in the use of chromatography making it unsuitable for purifying large quantities of oligonucleotide. The use of chromatography at large scale is expensive and is difficult to achieve due to the limitations on column size and performance.   2) The number of errors accumulates with the length of the oligonucleotide being synthesized. Accordingly, the linear sequential nature of the current process results in a geometric decrease in yield. For example, if the yield for each cycle of nucleotide addition is 99% then the yield of a 20 mer would be 83%.   3) Scale limitations with synthetic oligonucleotide synthesizers and downstream purification and isolation equipment: at present the maximum amount of product that can be produced in a single batch is in the order of 10 kg.       

     There is a need, therefore, to both reduce (or ideally eliminate) column chromatography and perform the synthesis in a way which is not purely sequential in order to increase yield. 
     DNA polymerase is often used to synthesize oligonucleotides for use in molecular biology and similar applications. However, DNA polymerase is unsuitable for synthesizing therapeutic oligonucleotides because of both the relatively short lengths of the oligonucleotides and the need to discriminate between nucleotides with different deoxyribose or ribose modifications. For example, therapeutic oligonucleotides are often in the range of 20 to 25 nucleotides. DNA polymerase needs at least 7 or 8 nucleotides, and optimally 18 to 22 nucleotides, as a primer in each direction so there is little to be gained in trying to synthesize a therapeutic oligo if the primers are similar in size to the desired product. Also, DNA polymerase requires all nucleotides to be present in the reaction and it relies on Watson-Crick base pairing to align incoming nucleotides. Thus it is unable to discriminate between any ordering of deoxyribose or ribose modifications, such as those required by a gapmer, and the result would be a mix of deoxyribose or ribose modifications at a given position. 
     SUMMARY OF THE INVENTION 
     The invention provides a process for producing a single stranded oligonucleotide product having at least one modified nucleotide residue, comprising:
         a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;   b) providing a pool of oligonucleotides (II);   c) contacting (I) and (II) in conditions to allow annealing;   d) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and   e) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.
 
Such a process may be used to isolate a single stranded oligonucleotide product from impurities, e.g. as a purification process.
       

     The invention further provides a process for producing a single stranded oligonucleotide product having at least one modified nucleotide residue, comprising:
         a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;   b) providing a pool of oligonucleotides (II) containing oligonucleotides that are segments of the product sequence, wherein at least one segment contains at least one modified nucleotide residue;   c) contacting (I) and (II) in conditions to allow annealing;   d) joining the segment oligonucleotides to form the product;   e) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and   f) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.
 
Such a process may be used to generate a single stranded oligonucleotide product and isolate it from impurities, e.g. as a manufacturing and purification process.
       

     The invention also encompasses modified oligonucleotides made by such methods and ligases for use in such methods. 
    
    
     
       DESCRIPTION OF FIGURES 
         FIG. 1  Schematic of the process of the invention, including a step of changing the conditions to remove impurities, e.g. a washing step. 
         FIG. 2  Schematic of the process of the invention, including the steps of joining/ligating the segment oligonucleotides to form the product and changing the conditions to remove impurities. 
         FIG. 3  Schematic of multiple template configurations. 
         FIG. 4  Basic schematic of the process of the invention being carried out in a flow system. 
         FIG. 5A ,  FIG. 5B ,  FIG. 5C  and  FIG. 5D  Detailed schematic of the process of the invention being carried out in a flow system: ligation chemistry section ( FIG. 5A ), ligation purification section ( FIG. 5B ), and alternative ligation chemistry section (FIG.  5 C), and alternative purification section ( FIG. 5D ). N.B. sections  FIG. 5A  and  FIG. 5B  (alternatively  FIG. 5C  and  FIG. 5D ) can be performed in a single step e.g. collection vessel in  FIG. 5A =output from ligation step in  FIG. 5B . 
         FIG. 6  Examples of impurities which may be generated during the process of the invention. 
         FIG. 7  Chromatogram showing the results of a ligation reaction using commercial NEB T4 ligase (SEQ ID NO:3) and unmodified DNA (Example 1). 
         FIG. 8  Chromatogram showing the results of a ligation reaction using PERLOZA bound T4 ligase (SEQ ID NO:4) and unmodified DNA (Example 1). 
         FIG. 9  Chromatogram showing the results of a ligation reaction using PERLOZA bound T4 ligase expressed according to Example 2 and 2′-OMe modified oligonucleotide fragments. 
         FIG. 10A  and  FIG. 10B  HPLC traces for Example 4: Upper trace ( FIG. 10A )—No ligase control. Lower trace ( FIG. 10B )—clone A4. Product and template co-elute in this HPLC method. Two intermediate ligation fragments (segments) can be seen in the Clone A4 trace at 10.3 and 11.2 minutes. 
         FIG. 11  Schematic of the “tri-template hub” used in Example 13, comprising a support material referred to as the “hub” and three template sequences. 
         FIG. 12  Schematic of the semi-continuous ligation rig used in Example 13. 
         FIG. 13  Schematic of the dead-end filtration rig used in Example 14. 
         FIG. 14  Schematic of the cross-flow filtration rig used in Example 14. 
         FIG. 15A  and  FIG. 15B  Chromatograms showing the results from the dead end filtration experiments using the 10 kDa MWCO NADIR membrane at 60° C. in Example 14.  FIG. 15A  is a chromatogram of the retentate solution, which remained in the filtration cell and contained mainly tri-template hub, after two diafiltration volumes; and chromatogram  FIG. 15B  is of the permeate, solution enriched in the product, after two diafiltration volumes. 
         FIG. 16A  and  FIG. 16B  Chromatograms showing the results from the cross-flow filtration experiments using the 5 kDa MWCO Snyder membrane at 50° C. and 3.0 bar pressure, in Example 14.  FIG. 16A  is a chromatogram of the retentate solution, which contained mainly tri-template hub and product, after 20 diafiltration volumes; and  FIG. 16B  is a chromatogram of the permeate, which contained mainly segment oligonucleotides, after 20 diafiltration volumes. 
         FIG. 17A  and  FIG. 17B  Chromatograms showing the results from the cross-flow filtration experiments using the 5 kDaMWCO Snyder membrane at 80° C. and 3.1 bar pressure, in Example 14.  FIG. 17A  shows a chromatogram of the retentate solution, which contained tri-template hub only, after 20 diafiltration volumes; and  FIG. 17B  is a chromatogram of the permeate solution, which contained the product only, after 2 diafiltration volumes. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     As used herein, the term “oligonucleotide”, or “oligo” for short, means a polymer of nucleotide residues, either deoxyribonucleotides (wherein the resulting oligonucleotide is DNA), ribonucleotides (wherein the resulting oligonucleotide is RNA), or a mixture thereof. An oligonucleotide may be entirely composed of nucleotide residues as found in nature or may contain at least one nucleotide, or at least one linkage between nucleotides, that has been modified. Oligonucleotides can be single stranded or double stranded. An oligonucleotide of the invention may be conjugated to another molecule, e.g. N-Acetylgalactosamine (GalNAc) or multiples thereof (GalNAc clusters). 
     As used herein, the term “therapeutic oligonucleotide” means an oligonucleotide that has a therapeutic application. Such an oligonucleotide typically contains one or more modified nucleotide residues or linkages. Therapeutic oligonucleotides act via one of several different mechanisms, including, but not limited to, antisense, splice-switching or exon-skipping, immunostimulation and RNA interference (RNAi), e.g. via microRNA (miRNA) and small interfering RNA (siRNA). A therapeutic oligonucleotide may be an aptamer. Therapeutic oligonucleotides will usually, but not always, have a defined sequence. 
     As used herein, the term “template” means an oligonucleotide with a sequence that is 100% complementary to the sequence of the target (or product) oligonucleotide. 
     Unless otherwise specified, as used herein, the term “complementary” means 100% complementary. 
     As used herein, the term “product” means the desired oligonucleotide, having a specific sequence, also referred to herein as a “target oligonucleotide”. 
     As used herein, the term “pool” refers to a group of oligonucleotides that may vary in sequence, may be shorter or longer than the target sequence, and may not have the same sequence as the target sequence. The pool of oligonucleotides may be the product of oligonucleotide synthesis, e.g. solid phase chemical synthesis via phosphoramidite chemistry, used with or without purification. The pool of oligonucleotides may be composed of segments of the target sequence. Each segment itself may be present as a pool of that segment and may be the product of oligonucleotide synthesis, e.g. solid phase chemical synthesis via phosphoramidite chemistry. 
     As used herein, the term “annealing” means the hybridisation of complementary oligonucleotides in a sequence specific manner. “Conditions to allow for annealing” will depend on the T m  of the hybridised complementary oligonucleotides and will be readily apparent to a person skilled in the art. For example, the temperature for annealing may be below the T m  of the hybridised oligonucleotides. Alternatively, the temperature for annealing may be close to the T m  of the hybridised oligonucleotides, e.g. +/−1, 2 or 3° C. The temperature for annealing is, in general, not higher than 10° C. above the T m  of the hybridised oligonucleotides. Specific conditions to allow for annealing are as outlined in the examples section. 
     As used herein, the term “denaturing” in relation to a double stranded oligonucleotide is used to mean that the complementary strands are no longer annealed. Denaturing occurs as a result of changing the conditions and is sometimes referred to herein as separating the oligonucleotide strands. Such strand separation can be done for example, by raising the temperature, changing the pH, or changing the salt concentration of the buffering solution. Denaturing a double stranded oligonucleotide (a “duplex”) results in a single stranded oligonucleotide which could be a product or impurity being “released” from the template. 
     As used herein, the term “impurity” or “impurities” means oligonucleotides that do not have the desired product sequence. These oligonucleotides may include oligonucleotides that are shorter than the product (for example 1, 2, 3, 4, 5 or more nucleotide residues shorter), or that are longer than the product (for example 1, 2, 3, 4, 5 or more nucleotide residues longer). Where the production process includes a step whereby linkages are formed between segments, impurities include oligonucleotides that are remaining if one or more of the linkages fail to form. Impurities also include oligonucleotides where incorrect nucleotides have been incorporated, resulting in a mis-match when compared to the template. An impurity may have one or more of the characteristics described above.  FIG. 6  shows some of the impurities that may occur. 
     As used herein, the term “segment” is a smaller portion of a longer oligonucleotide, in particular a smaller portion of a product or target oligonucleotide. For a given product, when all of its segments are annealed to its template and ligated together, the product is formed. 
     As used herein, the term “enzymatic ligation” means that the link between two adjacent nucleotides is formed enzymatically. This linkage may be a naturally occurring phosphodiester bond (PO), or a modified linkage including, but not limited to, phosphorothioate (PS) or phosphoramidate (PA). 
     As used herein, the term “ligase” means an enzyme that catalyses the joining, i.e. covalent joining, of two oligonucleotide molecules, e.g. by formation of a phosphodiester bond between the 3′ end of one oligonucleotide (or segment) and the 5′ end of the same or another oligonucleotide (or segment). These enzymes are often referred to as DNA ligases or RNA ligases and utilise cofactors: ATP (eukaryotic, viral and archael DNA ligases) or NAD (prokaryotic DNA ligases). Despite their occurrence in all organisms, DNA ligases show a wide diversity of amino acid sequences, molecular sizes and properties (Nucleic Acids Research, 2000, Vol. 28, No. 21, 4051-4058). They are usually members of the Enzyme Class EC 6.5 as defined by the International Union of Biochemistry and Molecular Biology, i.e. ligases used to form phosphoric ester bonds. Within the scope of the invention is a ligase capable of joining an unmodified oligonucleotide to another unmodified oligonucleotide, a ligase capable of joining an unmodified oligonucleotide to a modified oligonucleotide (i.e. a modified 5′ oligonucleotide to an unmodified 3′ oligonucleotide, and an unmodified 5′ oligonucleotide to a modified 3′ oligonucleotide), as well as a ligase capable of joining a modified oligonucleotide to another modified oligonucleotide. 
     As used herein, a “thermostable ligase” is a ligase that is active at elevated temperatures, i.e. above human body temperature, i.e. above 37° C. A thermostable ligase may be active at, for example, 40° C.-65° C.; or 40° C.-90° C.; and so forth. 
     As used herein, the term “modified nucleotide residue” or “modified oligonucleotide” means a nucleotide residue or oligonucleotide which contains at least one aspect of its chemistry that differs from a naturally occurring nucleotide residue or oligonucleotide. Such modifications can occur in any part of the nucleotide residue, e.g. sugar, base or phosphate. Examples of modifications of nucleotides are disclosed below. 
     As used herein, the term “modified ligase” means a ligase which differs from a naturally occurring, “wild-type”, ligase by one or more amino acid residues. Such ligases are not found in nature. Such ligases are useful in the novel processes of the invention. Examples of modified ligases are disclosed below. The terms “modified ligase” and “mutant ligase” are used interchangeably. 
     As used herein, the term “gapmer” means an oligonucleotide having an internal “gap segment” flanked by two external “wing segments”, wherein the gap segment consists of a plurality of nucleotides that support RNase H cleavage and each wing segment consists of one or more nucleotides that are chemically distinct to the nucleotides within the gap segment. 
     As used herein, the term “support material” means a high molecular weight compound or material that increases the molecular weight of the template, thereby allowing it to be retained when the impurities and products are separated from the reaction mixture. 
     As used herein “Percent identity” between a query nucleic acid sequence and a subject nucleic acid sequence is the “Identities” value, expressed as a percentage, that is calculated by the BLASTN algorithm when a subject nucleic acid sequence has 100% query coverage with a query nucleic acid sequence after a pair-wise BLASTN alignment is performed. Such pair-wise BLASTN alignments between a query nucleic acid sequence and a subject nucleic acid sequence are performed by using the default settings of the BLASTN algorithm available on the National Center for Biotechnology Institute&#39;s website with the filter for low complexity regions turned off. Importantly, a query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein. The query sequence may be 100% identical to the subject sequence, or it may include up to a certain integer number of nucleotide alterations as compared to the subject sequence such that the % identity is less than 100%. For example, the query sequence is at least 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. 
     STATEMENT OF THE INVENTION 
     In one aspect of the invention, there is provided a process for producing a single stranded oligonucleotide product having at least one modified nucleotide residue, comprising:
         a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;   b) providing a pool of oligonucleotides (II);   c) contacting (I) and (II) in conditions to allow annealing;   d) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and   e) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.       

     Such a process may be used to purify the product from impurities, e.g. a pool of oligonucleotides produced by chemical synthesis via phosphoramidite chemistry, e.g. solid phase chemical synthesis via phosphoramidite chemistry. 
     In a further embodiment of the invention there is provided a process comprising:
         a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product;   b) providing a pool of oligonucleotides (II) that contains short oligonucleotides which are segments of the target sequence, wherein at least one segment contains at least one modified nucleotide residue;   c) contacting (I) and (II) in conditions to allow annealing;   d) joining the segment oligonucleotides;   e) changing the conditions to separate any impurities, comprising denaturing the annealed template and impurity oligonucleotide strands and separating the impurities; and   f) changing the conditions to separate the product, comprising denaturing the annealed template and product oligonucleotide strands and separating the product.       

     In an embodiment of the invention, there are substantially no nucleotides in the reaction vessel. In an embodiment of the invention, there are no nucleotides in the reaction vessel. In particular, the reaction vessel does not comprise a pool of nucleotides, i.e. the reaction is substantially free, preferably free, of nucleotides. 
     Another embodiment of the invention provides a process as previously described herein, whereby the denaturation results from a temperature increase. 
     One embodiment of the invention provides a process as previously disclosed herein, wherein the segment oligonucleotides are joined by enzymatic ligation. In a further embodiment the enzymatic ligation is carried out by a ligase. 
     Another embodiment of the invention provides a process as previously disclosed herein, whereby the segment oligonucleotides are joined by chemical ligation. In a further embodiment, the chemical ligation is a click chemistry reaction. In an embodiment of the invention, chemical ligation of segment oligonucleotides takes place in templated reactions that produce phosphoramidate linkages as detailed by Kalinowski et al. in Chem Bio Chem 2016, 17, 1150-1155. 
     Yet another embodiment of the invention provides a process as previously disclosed herein, whereby the segment oligonucleotides are 3 to 15 nucleotides long. In a further embodiment of the invention the segments are 5 to 10 nucleotides long. In a further embodiment of the invention the segments are 5 to 8 nucleotides long. In a further embodiment of the invention the segments are 5, 6, 7 or 8 nucleotides long. In a particular embodiment there are three segment oligonucleotides: a 5′ segment that is 7 nucleotides long, a central segment that is 6 nucleotides long and a 3′ segment that is 7 nucleotides long, which when ligated together form an oligonucleotide that is 20 nucleotides long (a “20-mer”). In a particular embodiment there are three segment oligonucleotides: a 5′ segment that is 6 nucleotides long, a central segment that is 8 nucleotides long and a 3′ segment that is 6 nucleotides long, which when ligated together form an oligonucleotide that is 20 nucleotides long (a “20-mer”). In a particular embodiment there are three segment oligonucleotides: a 5′ segment that is 5 nucleotides long, a central segment that is 10 nucleotides long and a 3′ segment that is 5 nucleotides long, which when ligated together form an oligonucleotide that is 20 nucleotides long (a “20-mer”). In a particular embodiment there are four segment oligonucleotides: a 5′ segment that is 5 nucleotides long, a 5′-central segment that is 5 nucleotides long, a central-3′ segment that is 5 nucleotides long, and a 3′ segment that is 5 nucleotides long, which when ligated together form an oligonucleotide that is 20 nucleotides long (a “20-mer”). 
     One embodiment of the invention provides a process as previously described herein, whereby the product is 10 to 200 nucleotides long. In a further embodiment of the invention the product is 15 to 30 nucleotides long. In an embodiment of the invention the product is 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides long. In an embodiment of the invention the product is 20 nucleotides long, a “20-mer”. In an embodiment of the invention the product is 21 nucleotides long, a “21-mer”. In an embodiment of the invention the product is 22 nucleotides long, a “22-mer”. In an embodiment of the invention the product is 23 nucleotides long, a “23-mer”. In an embodiment of the invention the product is 24 nucleotides long, a “24-mer”. In an embodiment of the invention the product is 25 nucleotides long, a “25-mer”. In an embodiment of the invention the product is 26 nucleotides long, a “26-mer”. In an embodiment of the invention the product is 27 nucleotides long, a “27-mer”. In an embodiment of the invention the product is 28 nucleotides long, a “28-mer”. In an embodiment of the invention the product is 29 nucleotides long, a “29-mer”. In an embodiment of the invention the product is 30 nucleotides long, a “30-mer”. 
     In an embodiment of the invention, the process is a process for producing a therapeutic oligonucleotide. In an embodiment of the invention, the process is a process for producing a single stranded therapeutic oligonucleotide. In an embodiment of the invention, the process is a process for producing a double stranded therapeutic oligonucleotide. 
     Another embodiment of the invention provides a process as previously disclosed herein, wherein the property that allows the template to be separated from the product is that the template is attached to a support material. In a further embodiment of the invention, the support material is a soluble support material. In a yet further embodiment of the invention the soluble support material is selected from the group consisting of: polyethylene glycol, a soluble organic polymer, DNA, a protein, a dendrimer, a polysaccharide, an oligosaccharide, and a carbohydrate. In an embodiment of the invention the support material is polyethylene glycol (PEG). In a further embodiment of the invention, the support material is an insoluble support material. In a further embodiment of the invention the support material is a solid support material. In a yet further embodiment, the solid support material is selected from the group consisting of: a glass bead, a polymeric bead, a fibrous support, a membrane, a streptavidin coated bead and cellulose. In an embodiment the solid support material is a streptavidin coated bead. In a further embodiment, the solid support material is part of the reaction vessel itself, e.g. a reaction wall. 
     One embodiment of the invention provides a process as previously disclosed herein, wherein multiple, repeated copies of the template are attached in a continuous manner via a single attachment point to the support material. The multiple repeated copies of the template may be separated by a linker, e.g. as shown in  FIG. 3 . The multiple repeated copies of the template may be direct repeats, e.g. they are not separated by a linker. 
     Yet another embodiment of the invention provides a process as previously disclosed herein, wherein the property that allows the template to be separated from the product is the molecular weight of the template. For example, repeated copies of the template sequence may be present on a single oligonucleotide, with or without a linker sequence. 
     Another embodiment of the invention provides a process as previously disclosed herein, wherein the template, or the template and support material, are recycled for use in future reactions, for example as detailed below. Another embodiment of the invention provides a process as previously disclosed herein, wherein the reaction is carried out using a continuous or semi-continuous flow process, for example as shown in  FIG. 4  or  FIGS. 5A, 5B, 5C and 5D . 
     In an embodiment of the invention, the process is for large scale manufacture of oligonucleotides, in particular therapeutic oligonucleotides. In the context of the present invention large scale manufacture of oligonucleotides means manufacture at a scale greater than or equal to 1 litre, e.g. the process is carried out in a 1 L or larger reactor. Alternatively or in addition, in the context of the present invention large scale manufacture of oligonucleotides means manufacture at gram scale of product, in particular the production of greater than or equal to 10 grams of product. In an embodiment of the invention, the amount of oligonucleotide product produced is at gram scale. In an embodiment of the invention the amount of product produced is greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 grams. In an embodiment of the invention, the amount of oligonucleotide product produced is greater than or equal to: 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 grams. In an embodiment of the invention, the amount of oligonucleotide product produced is 500 grams or greater. In an embodiment of the invention, the oligonucleotide product produced is at kilogram scale. In an embodiment of the invention, the amount of oligonucleotide product produced is 1 kg or more. In an embodiment of the invention, the amount of oligonucleotide product produced is greater than or equal to: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 kg. In an embodiment of the invention, the amount of oligonucleotide product produced is greater than or equal to: 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kg. 
     In an embodiment of the invention, the amount of product produced is between 10 grams and 100 kg. In an embodiment of the invention, the amount of product produced is between 10 grams and 50 kg. In an embodiment of the invention, the amount of product produced is between 100 grams and 100 kg. In an embodiment of the invention, the amount of product produced is between 100 grams and 50 kg. In an embodiment of the invention, the amount of product produced is between 500 grams and 100 kg. In an embodiment of the invention, the amount of product produced is between 500 grams and 50 kg. In an embodiment of the invention, the amount of product produced is between 1 kg and 50 kg. In an embodiment of the invention, the amount of product produced is between 10 kg and 50 kg. 
     In an embodiment of the invention, oligonucleotide manufacture takes place at a scale greater than or equal to: 2, 3, 4, 5, 6, 7, 8, 9, 10 litres, e.g. in a 2, 3, 4, 5, 6, 7, 8, 9 or 10 L reactor. In an embodiment of the invention, oligonucleotide manufacture takes place at a scale greater than or equal to: 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 litres, e.g. in a 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 100 L reactor. In an embodiment of the invention, oligonucleotide manufacture takes place at a scale greater than or equal to: 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 litres, e.g. in 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 L reactor. 
     In an embodiment of the invention, the reactor volume is about 10,000 L, about 5000 L, about 2000 L, about 1000 L, about 500 L, about 125 L, about 50 L, about 20 L, about 10 L, or about 5 L. 
     In an embodiment of the invention, the reactor volume is between 5 and 10,000 L, between 10 and 5000 L, between 20 and 2000 L, or between 50 and 1000 L. 
     An oligonucleotide in accordance with the present invention may have at least one backbone modification, and/or at least one sugar modification and/or at least one base modification compared to an RNA or DNA-based oligonucleotide. 
     One embodiment of the invention provides a process as previously disclosed herein, wherein the product contains at least 1 modified nucleotide residue. In a further embodiment the modification is at the 2′ position of the sugar moiety. 
     Oligonucleotides used in the process of the invention may include sugar modifications, i.e. a modified version of the ribosyl moiety, such as 2′-O-modified RNA such as 2′-O-alkyl or 2′-O-(substituted)alkyl e.g. 2′-O-methyl, 2′-O-(2-cyanoethyl), 2′-O-(2-methoxy)ethyl (2′-MOE), 2′-O-(2-thiomethyl)ethyl, 2′-O-butyryl, 2′-O-propargyl, 2′-O-allyl, 2′-O-(3-amino)propyl, 2′-O-(3-(dimethylamino)propyl), 2′-O-(2-amino)ethyl, 2′-O-(2-(dimethylamino)ethyl); 2′-deoxy (DNA); 2′-O-(haloalkoxy)methyl (Arai K. et al. Bioorg. Med. Chem. 2011, 21, 6285) e.g. 2′-O-(2-chloroethoxy)methyl (MCEM), 2′-O-(2,2-dichloroethoxy)methyl (DCEM); 2′-O-alkoxycarbonyl e.g. 2′-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2′-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2′-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2′-halo e.g. 2′-F, FANA (2′-F arabinosyl nucleic acid); carbasugar and azasugar modifications; 3′-O-alkyl e.g. 3′-O-methyl, 3′-O-butyryl, 3′-O-propargyl; and their derivatives. 
     In an embodiment of the invention, the sugar modification is selected from the group consisting of 2′-Fluoro (2′-F), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), and 2′-amino. In a yet further embodiment, the modification is 2′-MOE. 
     Other sugar modifications include “bridged” or “bicylic” nucleic acid (BNA), e.g. locked nucleic acid (LNA), xylo-LNA, α-L-LNA, β-D-LNA, cEt (2′-O,4′-C constrained ethyl) LNA, cMOEt (2′-O,4′-C constrained methoxyethyl) LNA, ethylene-bridged nucleic acid (ENA), tricyclo DNA; unlocked nucleic acid (UNA); cyclohexenyl nucleic acid (CeNA), altriol nucleic acid (ANA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3′-deoxypyranosyl-DNA (p-DNA); morpholino (as e.g. in PMO, PPMO, PMOPlus, PMO-X); and their derivatives. 
     Oligonucleotides used in the process of the invention may include other modifications, such as peptide-base nucleic acid (PNA), boron modified PNA, pyrrolidine-based oxy-peptide nucleic acid (POPNA), glycol- or glycerol-based nucleic acid (GNA), threose-based nucleic acid (TNA), acyclic threoninol-based nucleic acid (aTNA), oligonucleotides with integrated bases and backbones (ONIBs), pyrrolidine-amide oligonucleotides (POMs); and their derivatives. 
     In an embodiment of the invention, the modified oligonucleotide comprises a phosphorodiamidate morpholino oligomer (PMO), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a bridged nucleic acid (BNA) such as (S)-cEt-BNA, or a SPIEGELMER. 
     In a further embodiment, the modification is in the nucleobase. Base modifications include modified versions of the natural purine and pyrimidine bases (e.g. adenine, uracil, guanine, cytosine, and thymine), such as inosine, hypoxanthine, orotic acid, agmatidine, lysidine, 2-thiopyrimidine (e.g. 2-thiouracil, 2-thiothymine), G-clamp and its derivatives, 5-substituted pyrimidine (e.g. 5-methylcytosine, 5-methyluracil, 5-halouracil, 5-propynyluracil, 5-propynylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5-hydroxymethylcytosine, Super T), 2,6-diaminopurine, 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza-7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, and N4-ethylcytosine, or derivatives thereof; N 2 -cyclopentylguanine (cPent-G), N 2 -cyclopentyl-2-aminopurine (cPent-AP), and N 2 -propyl-2-aminopurine (Pr-AP), or derivatives thereof; and degenerate or universal bases, like 2,6-difluorotoluene or absent bases like abasic sites (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the ring oxygen has been replaced with nitrogen (azaribose)). Examples of derivatives of Super A, Super G and Super T can be found in U.S. Pat. No. 6,683,173. cPent-G, cPent-AP and Pr-AP were shown to reduce immunostimulatory effects when incorporated in siRNA (Peacock H. et al. J. Am. Chem. Soc. (2011), 133, 9200). 
     In an embodiment of the invention, the nucleobase modification is selected from the group consisting of 5-methyl pyrimidines, 7-deazaguanosines and abasic nucleotides. In an embodiment, the modification is a 5-methyl cytosine. 
     Oligonucleotides used in the process of this invention may include a backbone modification, e.g. a modified version of the phosphodiester present in RNA, such as phosphorothioate (PS), phosphorodithioate (PS2), phosphonoacetate (PACE), phosphonoacetamide (PACA), thiophosphonoacetate, thiophosphonoacetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl phosphonothioate, methyl phosphate, methyl phosphorothioate, ethyl phosphate, ethyl phosphorothioate, boranophosphate, boranophosphorothioate, methyl boranophosphate, methyl boranophosphorothioate, methyl boranophosphonate, methylboranophosphonothioate, and their derivatives. Another modification includes phosphoramidite, phosphoramidate, N3′→P5′ phosphoramidate, phosphordiamidate, phosphorothiodiamidate, sulfamate, dimethylenesulfoxide, sulfonate, triazole, oxalyl, carbamate, methyleneimino (MMI), and thioacetamido nucleic acid (TANA); and their derivatives. 
     In a further embodiment, the modification is in the backbone and is selected from the group consisting of: phosphorothioate (PS), phosphoramidate (PA) and phosphorodiamidate. In an embodiment of the invention, the modified oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). A PMO has a backbone of methylenemorpholine rings with phosphorodiamidate linkages. In an embodiment of the invention the product has a phosphorothioate (PS) backbone. 
     In an embodiment of the invention, the oligonucleotide comprises a combination of two or more modifications as disclosed above. A person skilled in the art will appreciate that there are many synthetic derivatives of oligonucleotides. 
     In an embodiment of the invention, the product is a gapmer. In an embodiment of the invention the 5′ and 3′ wings of the gapmer comprise or consist of 2′-MOE modified nucleotides. In an embodiment of the invention the gap segment of the gapmer comprises or consists of nucleotides containing hydrogen at the 2′ position of the sugar moiety, i.e. is DNA-like. In an embodiment of the invention the 5′ and 3′ wings of the gapmer consist of 2′MOE modified nucleotides and the gap segment of the gapmer consists of nucleotides containing hydrogen at the 2′ position of the sugar moiety (i.e. deoxynucleotides). In an embodiment of the invention the 5′ and 3′ wings of the gapmer consist of 2′MOE modified nucleotides and the gap segment of the gapmer consists of nucleotides containing hydrogen at the 2′ position of the sugar moiety (i.e. deoxynucleotides) and the linkages between all of the nucleotides are phosphorothioate linkages. 
     One embodiment of the invention provides a process as previously described herein, wherein the resulting product is greater than 90% pure. In a further embodiment, the product is greater than 95% pure. In a further embodiment, the product is greater than 96% pure. In a further embodiment, the product is greater than 97% pure. In a further embodiment, the product is greater than 98% pure. In a further embodiment, the product is greater than 99% pure. Purity of an oligonucleotide may be determined using any suitable method, e.g. high performance liquid chromatography (HPLC) or mass spectrometry (MS), in particular liquid chromatography-MS (LC-MS), HPLC-MS or capillary electrophoresis mass spectrometry (CEMS). 
     Yet another embodiment of the invention provides a process for producing double stranded oligonucleotides, wherein 2 complimentary single stranded oligonucleotides are produced by the method of any preceding embodiment and then mixed under conditions to allow annealing. In an embodiment, the product is a siRNA. 
     One embodiment of the invention provides an oligonucleotide produced by a process previously described herein. In an embodiment of the invention, the oligonucleotide produced is RNA. In an embodiment of the invention, the oligonucleotide produced is DNA. In an embodiment of the invention, the oligonucleotide produced comprises both RNA and DNA. In a further embodiment of the invention the oligonucleotide produced is a modified oligonucleotide. In an embodiment of the invention the oligonucleotide produced is an antisense oligonucleotide. In an embodiment of the invention the oligonucleotide produced is an aptamer. In an embodiment of the invention the oligonucleotide produced is a miRNA. In an embodiment of the invention, the product is a therapeutic oligonucleotide. 
     The invention herein disclosed utilises the properties of oligonucleotide binding to provide an improved process for their production. By providing a template oligonucleotide with 100% complementarity to the target sequence, and controlling the reaction conditions so that the product can be released and separated under specific conditions, a product with a high degree of purity can be obtained. 
     Releasing the Product (or Impurity) from the Template, i.e. Denaturing Product (or Impurity):Template Duplex, and Separating the Product (or Impurity) 
     Releasing the product (or where the process includes an additional step of impurity release, any impurities) from the template requires the Watson-Crick base pairing between the template oligonucleotide strand and the product (or impurity) to be broken. The product (or impurity) can then be separated from the template. This can occur as two separate steps, or as one combined step. 
     Releasing and separating the product (or impurity) can occur as one step, if the process is carried out in a column reactor. Running in a buffer that alters the pH or salt concentration, or contains a chemical agent that disrupts the base pairing (such as formamide or urea) will cause denaturation of the oligonucleotide strands, and the product (or impurity) will be eluted in the buffer. 
     When the process is carried out in other reaction vessels, the release and separation of the product (or impurity) can occur as a two-step process. First, the Watson-Crick base pairs are disrupted to separate the strands, and then the product (or impurity) is removed from the reaction vessel. When releasing and separating the product is carried out as a two-step process, the breaking of the Watson-Crick base pairs can be achieved by altering the buffer conditions (pH, salt) or introducing a chemical disrupting agent (formamide, urea). Alternatively, raising the temperature will also cause the dissociation of the two strands. The product (or impurities) can then be removed from the reaction vessel via methods including molecular weight based separation, charge based separation, hydrophobicity based separation, specific sequence based separation or a combination of these methods. 
     When the process is carried out in a continuous or semi-continuous flow reactor the release and separation of the product (or impurity) can be in either one step or two steps. For example, releasing and separating the product (or impurity) in one step could be effected by increasing the temperature to cause dissociation of the two strands and separating the released strands on the basis of molecular weight in the same part of the reactor that is used to elevate the temperature. Releasing and separating the product (or impurity) in two steps could be effected by increasing the temperature to cause dissociation of the two strands in one part of the reactor and separating the released strands on the basis of molecular weight in a different part of the reactor. 
     Specifically Releasing and Separating Impurities from the Template, but Retaining the Product on the Template 
     Impurities arise when an incorrect nucleotide is incorporated into the oligonucleotide strand during chain extension, or when the chain extension reaction terminates early. Impurities also arise when the reaction includes the step of ligating segment oligonucleotides and one or more of the ligation steps fail to happen. The kinds of impurities which can arise are illustrated in  FIG. 6 . 
     The properties of Watson-Crick base pairing can be exploited to specifically release any impurities bound to the template prior to the release of the product. Each double stranded oligonucleotide will dissociate under specific conditions, and those conditions are different for sequences which do not have 100% complementarity when compared to sequences with 100% complementarity. Determining such conditions is within the remit of a skilled person. 
     A common way of denaturing oligonucleotides is by raising the temperature. The temperature at which half of the base pairs are dissociated, i.e. when 50% of the duplex is in the single-stranded state, is called the melting temperature, T m . The most reliable and accurate means of determining the melting temperature is empirically. However, this is cumbersome and not usually necessary. Several formulas can be used to calculate T m  values (Nucleic Acids Research 1987, 15 (13): 5069-5083; PNAS 1986, 83 (11): 3746-3750;  Biopolymers  1997, 44 (3): 217-239) and numerous melting temperature calculators can be found on-line, hosted by reagent suppliers and universities. It is known that for a given oligonucleotide sequence, a variant with all phosphorothioate linkages will melt at a lower temperature than a variant with all phosphodiester linkages. Increasing the number of phosphorothioate linkages in an oligonucleotide tends to lower the T m  of the oligonucleotide for its intended target. 
     To specifically separate the impurities from a reaction mixture, first the melting temperature of the product:template duplex is calculated. Then the reaction vessel is heated to a first temperature, e.g. a temperature below the melting temperature of the product:template duplex, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 degrees centigrade below the melting temperature. This will cause the denaturing of oligonucleotides which are not the product, i.e. are not 100% complimentary to the template, from the template. These can then be removed from the reaction vessel using one of the methods disclosed above, e.g. molecular weight based separation, charge based separation, hydrophobicity based separation, specific sequence based separation or a combination of these methods. Then the reaction vessel will be raised to a second, higher, temperature, e.g. above the calculated melting temperature, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 degrees centigrade above the melting temperature, to cause the denaturing of the product from the template. The product can then be removed from the reaction vessel using one of the methods disclosed above, e.g. molecular weight based separation, charge based separation, hydrophobicity-based separation, specific sequence based separation or a combination of these methods. 
     A similar process can be used when the disrupting agent is an agent which causes a change in pH, salt concentration or a chemical disrupting agent. The disrupting agent is increased in concentration until just below the concentration at which the product would dissociate, to cause the denaturing of oligonucleotides which are not the product from the template. These impurities can then be removed from the reaction vessel using one of the methods disclosed above. The disrupting agent is then increased in concentration to above the concentration at which the product dissociates from the template. The product can then be removed from the reaction vessel using one of the methods disclosed above. 
     The product obtained from a process such as disclosed above has a high degree of purity without the need for further purification steps. For example, the product obtained is greater than 95% pure. 
     Properties of the Template 
     The template requires a property which allows it to be retained in the reaction vessel when the product is removed, to prevent it from becoming an impurity in the product. In other words, the template has properties that allow it to be separated from the product. In one embodiment of the invention, this retention is achieved by coupling the template to a supporting material. This coupling results in a template-support complex which has a high molecular weight, and can therefore be retained in the reaction vessel when impurities and product are removed, for example by filtration. The template can be coupled to a solid support material such as polymeric beads, fibrous supports, membranes, streptavidin coated beads and cellulose. The template can also be coupled to a soluble support material such as polyethylene glycol, a soluble organic polymer, DNA, a protein, a dendrimer, a polysaccharide, an oligosaccharide and a carbohydrate. 
     Each support material can have multiple points where a template can be attached, and each attachment point can have multiple templates attached, e.g. in the manner shown in  FIG. 3 . 
     The template may have a high molecular weight itself, without being attached to a support material, for example, it may be a molecule with multiple copies of the template, e.g. separated by a linker, in the manner shown in  FIG. 3 . 
     The ability to retain the template in the reaction vessel also allows the template to be recycled for future reactions, either by being recovered or by use in a continuous or semi-continuous flow process. 
     Methods of Separating the Template from the Product (or Impurities) 
     The properties of the template, as disclosed above, allow separation of the template and product, or separation of the template bound product and impurities. Molecular weight based separation, charge based separation, hydrophobicity based separation, specific sequence based separation or a combination of these methods can be used. 
     In the case where the template is attached to a solid support, separation of the template from the product or separation of impurities from the product bound to the template is achieved by washing the solid support under appropriate conditions. In cases where the template is coupled to a soluble support or is itself composed of repeating template sequences, separation of template from product or separation of template bound product from impurities can be achieved by means of a molecular weight based separation. This can be achieved by using techniques such as ultra-filtration or nano-filtration where the filter material is chosen so that the larger molecule is retained by the filter and the smaller molecule passes through. In cases where a single separation step of impurity from template product complex or separation of product from template is not efficient enough, multiple sequential filtration steps can be employed to increase separation efficiency and so generate a product that meets the desired purity. 
     It is desirable to provide a process for separation of such oligonucleotides which is efficient and applicable on an industrial production scale. “Therapeutic oligonucleotides: The state of the art in purification technologies” Sanghvi et. al. Current Opinion in Drug Discovery (2004) Vol. 7 No. 8 reviews processes used for oligonucleotide purification. 
     WO-A-01/55160 discloses purification of oligonucleotides by forming imine linkages with contaminants then removing the imine-linked impurities with chromatography or other techniques. “Size Fractionation of DNA Fragments Ranging from 20 to 30000 Base Pairs by Liquid/Liquid chromatography” Muller et al. Eur. J. Biochem (1982) 128-238 discloses use of a solid column of microcrystalline cellulose on which has been deposited a PEG/dextran phase for separation of nucleotide sequences. “Separation and identification of oligonucleotides by hydrophilic interaction chromatography.” Easter et. al. The Analyst (2010); 135(10) discloses separation of oligonucleotides using a variant of HPLC employing a solid silica support phase. “Fractionation of oligonucleotides of yeast soluble ribonucleic acids by countercurrent distribution” Doctor et al. Biochemistry (1965) 4(1) 49-54 discloses use of a dry solid column packed with dry DEAE-cellulose. “Oligonucleotide composition of a yeast lysine transfer ribonucleic acid” Madison et al; Biochemistry, 1974, 13(3) discloses use of solid phase chromatography for separation of nucleotide sequences. 
     Liquid-liquid chromatography is a known separation method. “Countercurrent Chromatography The Support-Free Liquid Stationary Phase” Billardello, B.; Berthod, A; Wilson &amp; Wilson&#39;s Comprehensive Analytical Chemistry 38; Berthod, A., Ed.; Elsevier Science B.V.: Amsterdam (2002) pp 177-200 provides a useful general description of liquid-liquid chromatography. Various liquid-liquid chromatography techniques are known. One technique is liquid-liquid counter current chromatography (termed herein “CCC”). Another known technique is centrifugal partition chromatography (termed herein “CPC”). 
     The above disclosed methods and those methods set out in WO 2013/030263 may be used to separate a product oligonucleotide e.g. from the template and/or an impurity. 
     Oligonucleotides Used as Starting Materials 
     The oligonucleotides used as a starting material for the processes of the invention are herein described as being a “pool” and a definition thereof is provided above. The pool is a non-homogenous set of oligonucleotides. The oligonucleotides which form the pool will have been produced by other oligonucleotide production methods, and will therefore likely contain a high degree of impurities. Therefore when this pool of oligonucleotides is applied to a process of the invention, the ability to specifically remove impurities as described herein results in a purification step occurring. 
     The pool can contain oligonucleotides which are intended to be the same length as the template oligonucleotide (although will contain impurities of differing lengths, as well as incorrectly incorporated residues). The pool can also be composed of segments of the product oligonucleotides, which are joined together whilst assembled on the template. Each segment will be a non-homogeneous set with impurities of differing lengths and incorrectly incorporated residues. 
     Lipases 
     In an aspect of the invention, a ligase is provided. In an embodiment of the invention, the ligase is an ATP dependent ligase. ATP dependent ligases range in size from 30 to &gt;100 kDa. In an embodiment of the invention, the ligase is an NAD dependent ligase. NAD dependent enzymes are highly homologous and are monomeric proteins of 70-80 kDa. In an embodiment of the invention, the ligase is a thermostable ligase. A thermostable ligase may be derived from a thermophilic bacteria. 
     In an embodiment of the invention, the ligase is a modified ligase. For example, a modified ligase includes a modified T4 DNA ligase, a modified Enterobacteria phage CC31 ligase, a modified Shigella phage Shf125875 ligase and a modified Chlorella ligase. 
     In an embodiment, wild-type T4 DNA ligase is modified at amino acid position 368 or amino acid position 371 of SEQ ID NO:3. 
     In an embodiment, the mutant ligase comprises or consists of SEQ ID NO:3 wherein the amino acid at position 368 is R or K. 
     In an embodiment, the mutant ligase comprises or consists of SEQ ID NO:3 wherein the amino acid at position 371 is any one of the following amino acids: L, K, Q, V, P, R. 
     In an embodiment, the corresponding residue(s) disclosed above in relation to T4 DNA ligase are mutated in any one of Enterobacteria phage CC31 ligase, Shigella phage Shf125875 ligase and Chlorella ligase. Conserved regions of DNA ligases are disclosed in Chem. Rev. 2006, 106, 687-699 and Nucleic Acids Research, 2000, Vol. 28, No. 21, 4051-4058. In an embodiment, the ligase is modified in a linker region. 
     In an embodiment of the invention, the ligase comprises or consists of SEQ ID NO:23 or a ligase with at least 90% sequence identity thereto, excluding a wild type ligase e.g. Enterobacteria phage CC31 ligase. 
     In an embodiment of the invention, the ligase comprises or consists of any one of the following amino acid sequences: SEQ ID NOs:10-28. 
     In an embodiment of the invention, the ligase is immobilised, e.g. on a bead. 
     In an aspect of the invention there is provided the use of a ligase comprising the amino acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8 for ligating a 5′ segment, containing one or more modified sugar moieties, to a 3′ segment, wherein all of the sugar moieties within the 3′ segment are unmodified. In an embodiment of the invention there is provided the use of a ligase comprising the amino acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8 for ligating a 5′ segment, containing one or more sugar moieties with a 2′-OMe modification, to a 3′ segment, wherein all of the sugar moieties within the 3′ segment are unmodified. In an embodiment, all of the sugar moieties in the 5′ segment contain a 2′-OMe modification. In an embodiment, the 5′ segment contains 5 sugar moieties with a 2′-OMe modification. 
     The present invention includes the following items: 
     1. A process for producing a single stranded oligonucleotide product comprising: 
     a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product; 
     b) providing a pool of oligonucleotides (II); 
     c) contacting (I) and (II) in conditions to allow annealing; and 
     d) changing the conditions to remove the product. 
     2. A process according to item 1, comprising: 
     a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product; 
     b) providing a pool of oligonucleotides (II); 
     c) contacting (I) and (II) in conditions to allow annealing; 
     d) changing the conditions to remove impurities; and 
     e) changing the conditions to remove the product. 
     3. A process according to item 1 or 2, comprising: 
     a) providing a template oligonucleotide (I) complimentary to the sequence of the product, said template having properties that allow it to be separated from the product; 
     b) providing a pool of oligonucleotides (II) containing oligonucleotides that are segments of the product sequence; 
     c) contacting (I) and (II) in conditions to allow annealing; 
     d) joining the segment oligonucleotides to form the product; 
     e) changing the conditions to remove impurities; and 
     f) changing the conditions to remove the product. 
     4. A process according to any preceding item, whereby the process takes place in a reaction vessel, and whereby changing the conditions to remove product comprises the step of separating the annealed oligonucleotide strands and the step of removing the product from the reaction vessel. 
     5. A process according to any one of items 2-4, whereby the process takes place in a reaction vessel, and whereby changing the conditions to remove impurities comprises the step of separating the annealed oligonucleotide strands and the step of removing the impurities from the reaction vessel. 
     6. A process according to item 4 or 5, whereby the strand separation results from a temperature increase. 
     7. A process according to item 6, including two steps of increasing the temperature: i) to separate annealed impurities and ii) to separate annealed product. 
     8. A process according to items 3-7, wherein the segment oligonucleotides are joined by enzymatic ligation. 
     9. The process according to item 8, wherein the enzyme is a ligase. 
     10. A process according to any one of items 3-9, wherein the segments are 3 to 15 nucleotides long. 
     11. A process according to any preceding item, wherein the product is 10 to 200 nucleotides long. 
     12. A process according to item 11, wherein the product is 20 to 25 nucleotides long. 
     13. A process according to item 12, comprising three segment oligonucleotides: a 5′ segment that is 7 nucleotides long, a central segment that is 6 nucleotides long and a 3′ segment that is 7 nucleotides long. 
     14. A process according to item 12, comprising three segment oligonucleotides: a 5′ segment that is 6 nucleotides long, a central segment that is 8 nucleotides long and a 3′ segment that is 6 nucleotides long. 
     15. A process according to item 12, comprising three segment oligonucleotides: a 5′ segment that is 5 nucleotides long, a central segment that is 10 nucleotides long and a 3′ segment that is 5 nucleotides long. 
     16. A process according to any preceding item, wherein the property that allows the template to be separated from the product is that the template is attached to a support material. 
     17. The process according to item 16 wherein the support material is a soluble support material. 
     18. A process according to item 17 wherein the support material is polyethylene glycol. 
     19. A process according to any one of items 11-18, wherein multiple, repeated copies of the template are attached in a continuous manner via a single attachment point to the support material. 
     20. A process according to any one of items 1-15, wherein the property that allows the template to be separated from the product is the molecular weight of the template. 
     21. A process according to any preceding item, wherein the template, or the template and support material, are recycled for use in future reactions. 
     22. A process according to any preceding item, wherein the reaction is carried out using a continuous flow process. 
     23. A process according to any preceding item wherein the product contains at least one modified nucleotide residue. 
     24. A process according to item 23, wherein at least one segment contains at least one modified nucleotide residue. 
     25. The process according to item 23 or item 24, wherein the modification is at the 2′ position of the sugar moiety. 
     26. The process according to item 25, wherein the modification is selected from the group consisting of 2′-F, 2′-OMe, 2′-MOE, and 2′-amino. 
     27. The process according to item 24, wherein the oligonucleotide comprises a PMO, a LNA, a PNA, a BNA, or a Spiegelmer™. 
     28. The process according to item 23 or item 24, wherein the modification is in the nucleobase. 
     29. The process according to item 28 wherein the modification is selected from the group consisting of 5-methyl pyrimidines, 7-deazaguanosines and abasic nucleotides. 
     30. The process according to item 23 or item 24, wherein the modification is in the backbone. 
     31. The process according to item 30 wherein the modification is selected from the group consisting of phosphorothioate, phosphoramidate and phosphorodiamidate. 
     32. A process according to any preceding item, wherein the resulting product is at least 90% pure. 
     33. The process according item 32, wherein the product is at least 95% pure. 
     34. A process for producing double stranded oligonucleotides, wherein 2 complimentary single stranded oligonucleotides are produced by the method of any preceding item and then mixed under conditions to allow annealing. 
     35. A process as claimed in any of the preceding items, wherein the process is for producing a therapeutic oligonucleotide. 
     36. An oligonucleotide produced by the process of any one of items 1 to 35. 
     37. An oligonucleotide according to claim  36 , wherein the oligonucleotide is a modified oligonucleotide produced by the process of any one of items 23 to 35. 
     38. An oligonucleotide according to item 37, wherein the oligonucleotide is a gapmer. 
     39. A ligase comprising the amino acid sequence as set out in SEQ ID NO:3 wherein the amino acid at position 368 is replaced with R or K; and/or the amino acid at position 371 is replaced with L, K, Q, V, P, or R. 
     40. A ligase comprising the amino acid sequence as set forth in any one of the following SEQ ID NOs:10-28. 
     41. Use of a ligase comprising the amino acid sequence as set forth in SEQ ID NO:6 or SEQ ID NO:8 for ligating a 5′ segment, containing one or more sugar moieties with a 2′-OMe modification, to a 3′ segment, wherein all of the sugar moieties within the 3′ segment are unmodified. 
     EXAMPLES 
     Abbreviations 
     
         
         OMe O-Methyl 
         MOE O-Methoxyethyl (DNA backbone) or Methoxyethyl (RNA backbone) 
         CBD Cellulose Binding Domain 
         HPLC high performance liquid chromatography 
         PBS phosphate buffered saline 
         HAA Hexylammonium acetate 
         SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis 
         LCMS liquid chromatography mass spectrometry 
         PO phosphodiester 
         DTT dithiothreitol 
       
    
     Example 1 
     Oligonucleotide (DNA) Segment Assembly and Ligation with Wild-Type T4 DNA Ligase 
     1.1 Chemical Synthesis of Starting and Control Sequences 
     In order to demonstrate that multiple short oligonucleotides (“segments”) could be assembled in the correct order on a complementary template strand and ligated to give the desired final product (“target”), the segments, target and template sequences, as detailed in Table 1, were chemically synthesised using standard methods. 
     1.2 HPLC Analysis 
     HPLC analysis was carried out using an Agilent ZORBAX Eclipse Plus XDB-C18 column (4.6×150 mm, 5 pm dp. Agilent P/N 993967-902) running at 0.2 ml/min while absorbance was monitored at 258 nm. The column was maintained at 60° C. 20 μl of sample was injected and a gradient from 20-31% buffer B was run over 20 minutes before being stepped up to 80% buffer B for 5 minutes. 
     Buffer A: 75 ml 1 M HAA, 300 ml isopropyl alcohol, 200 ml acetonitrile, 4425 ml water 
     Buffer B: 650 ml isopropyl alcohol, 350 ml acetonitrile 
                                 TABLE 1                       % HPLC   Amount       Name   Sequence   purity   (mg)                  5′ segment   5′-GGC CAA-3′   100.0    21.6               centre segment   5′-(p)ACC TCG GC-3′    96.9    58.1               3′ segment   5′-(p)T TAC CT-3′    98.8    39.8               Target   5′-GGC CAA ACC TCG GCT TAC    98.4   101.7           CT-3′ (SEQ ID NO: 1)                       Biotinylated   5′-biotin TT TAG GTA AGC CGA    96.9   130.7       template   GGT TTG GCC-3′ (SEQ ID NO: 2)                    
1.3 Oligonucleotide Assembly and Ligation Method with Commercial T4 DNA Ligase (SEQ ID NO:3)
 
     The 5′ segment, centre segment and 3′ segment were assembled on the template: each segment and the template was dissolved in water at a concentration of 1 mg/ml and then mixed as follows. 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 5′ segment 
                 2 μl 
               
               
                   
                 centre segment 
                 2 μl 
               
               
                   
                 3′ segment 
                 2 μl 
               
               
                   
                 biotinylated template 
                 2 μl 
               
               
                   
                 H 2 O 
                 36 μl  
               
               
                   
                   
               
            
           
         
       
     
     The combined oligonucleotide solution was incubated at 94° C. for 5 minutes and cooled to 37° C. before incubating at 37° C. for a further 5 minutes to allow the segments to anneal to the template. 2 μl (equivalent to 2 μg) T4 DNA ligase (NEB) and 4 μl of 1×T4 DNA Ligation Buffer (NEB) were then added and the reaction (total reaction volume 50 μl) was incubated at room temperature for one hour. Following this, 40 μl of streptavidin coated magnetic beads were added and the suspension incubated at room temperature for 10 minutes to allow the biotinylated template to bind to the streptavidin beads. The streptavidin beads were washed with 2×100 ∥l PBS to remove unbound segments. The wash was analysed by HPLC. The reaction mixture was then incubated at 94° C. for 10 minutes to separate the bound ligation products (or any bound segments) from the template before being rapidly cooled on ice to ‘melt’ the DNA and stop reannealing of the oligonucleotides products (or segments) to the template. Analysis of the ligation reaction was then carried out by HPLC. 
     1.4 Oligonucleotide Assembly and Ligation Method with In-House T4 DNA Ligase Bead Slurry 
     1.4.1 Bead Slurry Generation 
     T4 ligase (SEQ ID NO:4) fused at the N-terminal to a cellulose binding domain (CBD) was produced using standard cloning, expression and extraction methods. This T4 ligase amino acid sequence differs from the commercial T4 ligase sequence (SEQ ID NO:3) in that the N-terminal methionine (M) has been replaced with glycine and serine (GS). This was done to aid the generation and expression of the CBD fusion protein. CBD-T4 ligase fusion protein was expressed in BL21 A1 cells (INVITROGEN). Supernatent was harvested and was added to 600 μl of PERLOZA 100 (PERLOZA) beads and shaken at 26° C. for 1 hour. The PERLOZA cellulose beads were then collected and washed with 2 ml buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Tween 20, 10% Glycerol) followed by 5 ml PBS and were finally re-suspended in 200 μl PBS (10 mM PO 4   3− , 137 mM NaCl, 2.7 mM KCl pH 7.4). In order to analyse protein expression, 15 μl of the PERLOZA bead slurry was mixed with 5 μl of SDS loading buffer and incubated at 80° C. for 10 minutes before being run on a SDS PAGE gradient gel (4-20%) according to a standard protocol. 
     1.4.2 Oligonucleotide Assembly and Ligation Using Bead Slurry 
     For T4 ligase bound to PERLOZA beads, the assembly and ligation method in 1.3 above was modified as follows. In the initial segment mixture, 36 μl of H 2 O was reduced to 8 μl H 2 O. After annealing, 2 μl of commercial T4 DNA ligase was replaced by 20 μl of PERLOZA bead slurry. Prior to adding the streptavidin magnetic beads, the PERLOZA beads were spun down and the supernatant removed. The streptavidin magnetic beads were added to the supernatant and incubated at room temperature for 10 minutes to allow the biotinylated template to bind to the streptavidin beads. 
     1.5 Results and Conclusions 
     Product, template and all three segment oligonucleotides were clearly resolved in the control chromatogram. 
     HPLC analysis of the ligase reactions showed that some unligated oligonucleotide segments remained, but commercial T4 DNA ligase (NEB) was able to catalyse ligation of the three segments to generate the desired product oligonucleotide ( FIG. 7 ). The PERLOZA bead bound T4 DNA ligase appeared to be less efficient at ligation of the oligonucleotide segments, with oligonucleotide segments appearing in both the control wash sample and the reaction sample ( FIG. 8 ). However, it is difficult to be sure whether the same amount of enzyme was added on the beads compared to the commercial enzyme and so a direct comparison of ligation efficiency was not possible. 
     Example 2 
     2′-OMe Ribose Modified Oligonucleotide Segment Assembly and Ligation with Wild-Type T4 DNA Ligase 
     2.1 2′ OMe at Each Nucleotide Position in Every Segment 
     In order to determine whether T4 DNA ligase was able to ligate oligonucleotide segments with modification at the 2′ position of the ribose ring, oligonucleotide segments were synthesized with the same sequence as for Example 1, but the 2′ position of the ribose ring was substituted with an OMe group and thymidine was replaced by uridine as shown below. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                   
                 % HPLC 
                 Amount 
               
               
                 Name 
                 Sequence 
                 purity 
                 (mg) 
               
               
                   
               
             
            
               
                 5′ segment 2′-OMe 
                 5′-GGC CAA-3′ 
                 21 
                 97.8 
               
               
                   
               
               
                 centre segment 2′- 
                 5′-(p)ACC UCG GC-3′ 
                 15.5 
                 97.7 
               
               
                 OMe 
                   
                   
                   
               
               
                   
               
               
                 3′ segment 2′-OMe 
                 5′-(p)U UAC CU-3′ 
                 21.2 
                 98.1 
               
               
                   
               
               
                 Target 2′-OMe 
                 5′-GGC CAA ACC UCG GCU 
                 88 
                 96.9 
               
               
                   
                 UAC CU-3′ 
                   
                   
               
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
               
               
                 (p) = phosphate 
               
            
           
         
       
     
     Assembly, ligation and HPLC analysis were carried out using the methods of Example 1, with both commercial NEB ligase and T4 ligase CBD fusion bound to PERLOZA beads. The amount of water used in the reaction mix for the commercial T4 DNA ligase (NEB) experiment was 26 μl rather than 36 μl so that the final reaction volume was 40 μl. The amount of water used in the reaction mix for the in-house T4 DNA ligase bead slurry experiment was 23 μl and the amount of beads used was 5 μl so that the final reaction volume was also 40 μl. Control experiments using unmodified DNA as opposed to 2′-OMe DNA were run in parallel. 
     The results from the control experiments were in accordance with Example 1. No product was detected using HPLC for the 2′-OMe experiments indicating that T4 DNA ligase is unable to ligate fully 2′-OMe modified oligonucleotide segments regardless of whether a commercial T4 DNA ligase or in-house T4 DNA ligase CBD fusion bound to PERLOZA beads was used. 
     2.2 2′-OMe at Each Nucleotide Position in a Single Segment 
     Using a 1 mg/ml solution of each oligonucleotide the reactions as detailed in Table 3 were set up. 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                 Experiment 2 
                 Experiment 3 
                   
                   
                   
               
               
                 Experiment 1 
                 (single 2′- 
                 (single 2′- 
                   
                 Experiment 5 
               
               
                 (No ligase 
                 OMe 
                 OMe 
                 Experiment 4 
                 (all 
                 Volume 
               
               
                 control) 
                 segment - 3′) 
                 segment - 5′) 
                 (all 2′-OMe) 
                 unmodified) 
                 (μl) 
               
               
                   
               
             
            
               
                 template 
                 template 
                 template 
                 template 
                 template 
                 2 
               
               
                 5′ segment 
                 5′ segment 
                 2′-OMe 
                 2′-OMe 
                 5′ segment 
                 2 
               
               
                   
                   
                 substituted 
                 substituted 
               
               
                   
                   
                 5′ segment 
                 5′ segment 
               
               
                 3′ segment 
                 2′-OMe 
                 3′ segment 
                 2′-OMe 
                 3′ segment 
                 2 
               
               
                   
                 substituted 
                   
                 substituted 
               
               
                   
                 3′ segment 
                   
                 3′ segment 
               
               
                 Centre segment 
                 Centre segment 
                 Centre segment 
                 2′-OMe 
                 Centre segment 
                 2 
               
               
                   
                   
                   
                 substituted 
               
               
                   
                   
                   
                 Centre segment 
               
               
                 H 2 O 
                 H 2 O 
                 H 2 O 
                 H 2 O 
                 H 2 O 
                 up to 40 
               
               
                   
                   
                   
                   
                   
                 total 
               
               
                   
               
            
           
         
       
     
     Assembly and ligation were carried out using the methods of Example 1 with commercial NEB ligase and in-house PERLOZA bound T4 DNA ligase. 
     Reactions were incubated at 94° C. for 5 minutes, followed by incubation for 5 minutes at 37° C. to allow for annealing. 4 μl of 1× NEB T4 DNA ligation buffer was added to each reaction along with 5 μl (approximately 2 μg) of in-house T4 DNA ligase or 2 μl (approximately 2 μg) commercial T4 DNA ligase (apart from Experiment 1 which was a no ligase control) and the ligation reaction was allowed to proceed for 2 hours at room temperature. Streptavidin magnetic beads were then added to each reaction and the reactions heated to 94° C. before rapid cooling on ice as described in Example 1 to separate the template from starting materials and products. 
     The processed reactions were split in two: half were analysed by HPLC as described for Example 1 (section 1.2). The other half of the sample was retained for mass spectrometry to confirm the HPLC results. 
     Ligation of unmodified oligonucleotide segments (experiment 5) proceeded as expected to produce full length product. A small amount of ligation was seen when the 5′ segment was 2′-OMe substituted (Experiment 3) as shown in  FIG. 9 , but no ligation was seen when the 3′ segment was 2′-OMe substituted (Experiment 2). No significant product was seen when all three segments were 2′-OMe substituted (Experiment 4) in accordance with 2.1. 
     2.3 Conclusion 
     Wild-type T4 DNA ligase is poor at ligating 2′-OMe substituted oligonucleotide segments, but slightly less sensitive to modification of the 5′ oligonucleotide segment than the 3′ segment. 
     Example 3 
     2′-OMe Ribose Modified Oligonucleotide Segment Assembly and Ligation with Wild-Type and Mutant Ligases 
     3.1 Materials 
     Wild-type Enterobacteria phage ligase CC31 (SEQ ID NO:6), wild-type Shigella phage Shf125875 ligase (SEQ ID NO:8), and 10 mutant T4 ligases of SEQ ID NO:10-19, each fused at the N-terminus to a CBD, were produced using standard cloning, expression and extraction methods. As disclosed in 1.4.1, in order to generate and express the CBD fusion proteins the N-terminal methionine (M) was replaced with glycine and serine (GS) in each case (e.g. SEQ ID NO:7 for Enterobacteria phage ligase CC31 and SEQ ID NO:9 for Shigella phage Shf125875 ligase). 
     The following oligonucleotides were synthesized by standard solid phase methods. 
                                 TABLE 4                       % HPLC   Amount       Name   Sequence   purity   (mg)                  5′ segment *   5′-   99.35    24.7           (OMe)G(OMe)G(OMe)C(OMe)C(OMe)AA-                   3′                       centre segment   5′-(p)ACC TCG GC-3′   96.9    58.1               3′ segment   5′-(p)TTA CCT-3′   97.88    29.5               Biotinylated   5′-biotin TT TAG GTA AGC CGA GGT   96.9   130.7       template   TTG GCC-3′ (SEQ ID NO: 2)               N.B. OMe indicates 2′ methoxy substitution on the ribose ring       (p)= phosphate       * note that the first 5 nucleotides are 2′-OMe modified (GGCCA), but the final A is not            
3.2 Oligonucleotide Assembly and Ligation Method with Ligase Bead Slurries
 
     3.2.1 Bead Slurry Generation 
     Ligases fused to CBD were bound to PERLOZA beads as described in 1.4 to generate a bead slurry. 
     3.2.2 Oligonucleotide Assembly and Ligation Using Bead Slurry 
     Ligation reactions were prepared with the components below to a final volume of 50 μL in a 96 well plate: 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 2 μL 
                 ~1 mg/mL 5′ (2′-OMe) segment 
               
               
                 2 μL 
                 ~1 mg/mL centre segment 
               
               
                 2 μL 
                 ~1 mg/mL 3′ segment 
               
               
                 2 μL 
                 ~1 mg/mL template 
               
               
                 5 μL 
                 NEB T4 DNA ligase buffer 
               
               
                 22 μL  
                 H 2 O 
               
               
                 15 μL  
                 PERLOZA bound bead slurry 
               
               
                   
               
            
           
         
       
     
     The reaction was incubated for 15 minutes at room temperature prior to the addition of PERLOZA bead slurry to allow segments to anneal to the template. PERLOZA bead slurry was added and the reaction incubated at room temperature for 1 hour. After the hour incubation, the solution was transferred into an ACOPREP advance 350 filter plate (PN 8082) and the filter plate was placed on top of an ABGENE superplate (Thermo Scientific, #AB-2800) and centrifuged for 10 minutes at 4,000 rpm to remove the PERLOZA bead slurry. Solutions were then analysed by HPLC using the method described in Example 1 (section 1.2). 
     Each oligonucleotide assembly and ligation was repeated 6 times for each ligase. 
     3.3 Results and Conclusions 
     Wild-type Enterobacteria phage CC31 ligase (SEQ ID NO:6) and wild-type Shigella phage Shf125875 ligase (SEQ ID NO:8) are able to ligate a 2′ OMe substituted 5′ segment containing five 2′-OMe nucleobases and one deoxynucleobase to a segment containing only unmodified DNA. In addition, whilst wild-type T4 DNA ligase (SEQ ID NO:3 and 4) is poor at performing this reaction, as shown in Example 2 and reconfirmed here, a number of mutations at positions 368 and 371 confer the ability to ligate a 2′-OMe substituted 5′ segment containing five 2′ OMe nucleobases and one deoxynucleobase to a segment containing only unmodified DNA on the ligase (SEQ ID NO:10-19). 
     Example 4 
     2′ MOE Ribose Modified and 5-Methyl Pyrimidine Modified Oligonucleotide Segment Assembly and Ligation with Mutant DNA Ligases 
     4.1 Materials 
     Modified oligonucleotide segments as set out in Table 5 below were synthesised by standard solid phase based methods. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                   
                   
                   
                 Mass 
                 % 
               
               
                 Segment 
                 Sequence 
                 MW 
                 (mg) 
                 purity 
               
               
                   
               
             
            
               
                 centre segment 
                 5′-(p)dCdCdTdCdGdG-3′ 
                 2044.122 
                 39 
                 98.96 
               
               
                   
               
               
                 MOE 3′- 
                 5′- 
                 2699.679 
                 52 
                 97.79 
               
               
                 segment 
                 (p)dCdTmTmAmCmCmT-3′ 
                   
                   
                   
               
               
                   
               
               
                 MOE 5′- 
                 5′- 
                 2644.771 
                 48 
                 99.13 
               
               
                 segment 
                 mGmGmCmCmAdAdA-3′ 
               
               
                   
               
               
                 (p) = phosphate, mX = MOE bases, dX = DNA bases all 5-methyl pyrimidines 
               
            
           
         
       
     
     Mutant DNA ligases (SEQ ID NO:20-28) based upon wild type Enterobacteria phage CC31 ligase, wild type T4 ligase and wild type Shigella phage Shf125875 ligase were each fused at the N-terminus to a cellulose binding domain (CBD), using standard cloning, expression and extraction methods. Ligases fused to CBD were bound to PERLOZA beads as described in 1.4 to generate a bead slurry. In order to release the ligases from the PERLOZA beads, 2 μl of TEV protease was added to the slurry and incubated overnight at 4° C. The cleaved protein, now lacking the cellulose binding domain, was collected by centrifugation for 10 min at 4000 rpm. 
     4.2 Method 
     The reaction was set up as follows: 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 centre segment 
                 20 
                 μM final 
               
               
                 MOE 3′ segment 
                 20 
                 μM final 
               
               
                 MOE 5′ segment 
                 20 
                 μM final 
               
               
                 Template 
                 20 
                 μM final 
               
               
                 NEB T4 DNA ligase buffer 
                 5 
                 μl 
               
               
                 mutant DNA ligase 
                 15 
                 μl 
               
            
           
           
               
               
            
               
                 H 2 O 
                 To make final reaction volume 50 μl 
               
               
                   
               
            
           
         
       
     
     All components were mixed and vortexed prior to addition of DNA ligase. Reactions were incubated for 1 hour at 35° C. After 1 hour reactions were stopped by heating at 95° C. for 5 minutes in a PCR block. 
     Samples were analysed by both HPLC and LCMS to confirm product identity according to the HPLC protocol used in Example 1 (section 1.2). Controls of commercial NEB T4 DNA ligase and a negative control (H 2 O instead of any ligase) were included. 
     4.3 Results and Conclusions 
       FIG. 10A ,  FIG. 10B ,  FIG. 10C  and  FIG. 10D  shows the HPLC traces for the control reaction and the reaction catalysed by SEQ ID NO:23 (clone A4—mutant Enterobacteria phage CC31 ligase). The product and template co-elute using this HPLC method so product appears as an increase in the peak area of the product+template peak. In the mutant ligase trace (b) not only does the product+template peak increase but two new peaks appear at 10.3 and 11.2 minutes. These peaks correspond to the ligation of the centre segment with either the MOE 5′ segment or the MOE 3′ segment. Also, the input segment peaks are substantially smaller than the control in line with product and intermediate peaks increasing. The NEB commercial T4 ligase trace (a) showed a slight increase in the peak area for template+product, a small amount of intermediate ligation product along with a concomitant decrease in input oligonucleotide segments. The mutant ligase (SEQ ID NO:23), however, showed substantially greater product+template peak area and concomitant reduction in peak areas for input oligonucleotide segments. Thus the mutant ligase (SEQ ID NO:23) is a much more effective ligase for the 2′ MOE substituted segments than commercial T4 DNA ligase. Similar improvements were shown for the other mutant ligases (SEQ ID NO:20, 21, 24-28). 
     Example 5 
     Effect of Different Nucleotide Pairing at the Ligation Site 
     5.1 Materials 
     Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) fused at the N-terminal to a cellulose binding domain (CBD) was produced using standard cloning, expression and extraction methods. Extracted CBD-mutant Enterobacteria phage CC31 ligase fusion protein was added to 25 ml of PERLOZA 100 (PERLOZA) cellulose beads and shaken at 20° C. for 1 hour. The PERLOZA beads were then collected and washed with 250 ml buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Tween 20, 10% Glycerol) followed by 250 ml PBS and were finally re-suspended in 10 ml PBS (10 mM PO 4   3− , 137 mM NaCl, 2.7 mM KCl pH 7.4). In order to analyse protein expression, 15 μl of the PERLOZA bead slurry was mixed with 5 μl of SDS loading buffer and incubated at 80° C. for 10 minutes before being run on a SDS PAGE gradient gel (4-20%) according to a standard protocol. For the release of the ligase from the beads, 70 μl of TEV protease was added and incubated overnight at 4° C. with shaking. Ligase was collected by washing the digested beads with 80 ml of PBS. The ligase was then concentrated down to 1.2 ml using an Amicon 30 Kd MCO filter. 
     The following biotinylated DNA template oligonucleotides (Table 6) and DNA segment oligonucleotides (Table 7) were synthesized by standard solid phase methods. Please note that the nucleotides in bold are the ones present at the ligation site (i.e. those nucleotides that were joined together in the ligation reaction—table 7; and those nucleotides that are complementary to those joined via the ligation reaction—table 6). 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                 SEQ 
               
               
                 Template 
                 Sequence (nucleotides complementary 
                 ID 
               
               
                 number 
                 to junction nucleotides are in bold) 
                 NO 
               
               
                   
               
             
            
               
                  1 
                 5′-biotin TTTGGTGCGAAGCAG AC TGAGGC- 
                 30 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  2 
                 5′-biotin TTTGGTGCGAAGCAG AG TGAGGC- 
                 31 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  3 
                 5′-biotin TTTGGTGCGAAGCAG AT TGAGGC- 
                 32 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  4 
                 5′-biotin TTTGGTGCGAAGCAG AA TGAGGC- 
                 33 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  5 
                 5′-biotin TTTGGTGCGAAGCAG TC TGAGGC- 
                 34 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  6 
                 5′-biotin TTTGGTGCGAAGCAG TG TGAGGC- 
                 35 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  7 
                 5′-biotin TTTGGTGCGAAGCAG TT TGAGGC- 
                 36 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  8 
                 5′-biotin TTTGGTGCGAAGCAG TA TGAGGC- 
                 37 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                  9 
                 5′-biotin TTTGGTGCGAAGCAG CC TGAGGC- 
                 38 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 10 
                 5′-biotin TTTGGTGCGAAGCAG CG TGAGGC- 
                 39 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 11 
                 5′-biotin TTTGGTGCGAAGCAG CT TGAGGC- 
                 40 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 12 
                 5′-biotin TTTGGTGCGAAGCAG CA TGAGGC- 
                 41 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 13 
                 5′-biotin TTTGGTGCGAAGCAG GC TGAGGC- 
                 42 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 14 
                 5′-biotin TTTGGTGCGAAGCAG GG TGAGGC- 
                 43 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 15 
                 5′-biotin TTTGGTGCGAAGCAG GT TGAGGC- 
                 44 
               
               
                   
                 3′ 
                   
               
               
                   
               
               
                 16 
                 5′-biotin TTTGGTGCGAAGCAG GA TGAGGC- 
                 45 
               
               
                   
                 3′ 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Sequence (junction nucleotides 
               
               
                 Segment 
                 Identifier 
                 are in bold) 
               
               
                   
               
             
            
               
                 5′ 
                 A 
                 5′-GCCTCA G -3′ 
               
               
                   
               
               
                 5′ 
                 B 
                 5′-GCCTCA C -3′ 
               
               
                   
               
               
                 5′ 
                 C 
                 5′-GCCTCA A -3′ 
               
               
                   
               
               
                 5′ 
                 D 
                 5′-GCCTCA T -3′ 
               
               
                   
               
               
                 3′ 
                 E 
                 5′-(p) T CTGCT-3′ 
               
               
                   
               
               
                 3′ 
                 F 
                 5′-(p) A CTGCT-3′ 
               
               
                   
               
               
                 3′ 
                 G 
                 5′-(p) C CTGCT-3′ 
               
               
                   
               
               
                 3′ 
                 H 
                 5′-(p) G CTGCT-3′ 
               
               
                   
               
               
                 (p) = phosphate 
               
            
           
         
       
     
     N.B. please note that, unlike previous examples, the ligation reactions in this example involve joining two segments together: a 5′-segment and a 3′-segment, i.e. there is no centre segment. 
     5.2 Method 
     Reactions were set up as follows: 
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Template 
                 5′-segment 
                 3′-segment 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 A 
                 E 
               
               
                 2 
                 B 
                 E 
               
               
                 3 
                 C 
                 E 
               
               
                 4 
                 D 
                 E 
               
               
                 5 
                 A 
                 F 
               
               
                 6 
                 B 
                 F 
               
               
                 7 
                 C 
                 F 
               
               
                 8 
                 D 
                 F 
               
               
                 9 
                 A 
                 H 
               
               
                 10 
                 B 
                 H 
               
               
                 11 
                 C 
                 H 
               
               
                 12 
                 D 
                 H 
               
               
                 13 
                 A 
                 G 
               
               
                 14 
                 B 
                 G 
               
               
                 15 
                 C 
                 G 
               
               
                 16 
                 D 
                 G 
               
               
                   
               
            
           
         
       
     
     For each 50 μL reaction: 
     
       
         
           
               
               
             
               
                   
               
             
            
               
                 3′ segment (1 mM stock, 20 μM final) 
                 1 μl 
               
               
                 5′ segment (1 mM stock, 20 μM final) 
                 1 μl 
               
               
                 Template (1 mM stock, 20 μM final) 
                 1 μl 
               
               
                 NEB DNA ligase buffer (for T4 ligase) 
                 5 μl 
               
               
                 Mutant CC31 DNA ligase (0.45 mM stock, 90 μM final) 
                 10 μl  
               
               
                 H 2 O 
                 up to 50 μl 
               
               
                   
               
            
           
         
       
     
     Each reaction mix was incubated at 35° C. both for 30 minutes and 1 hour. Each reaction was terminated by heating at 95° C. for 5 minutes. HPLC analysis was carried out. 
     5.3 Results and Conclusions 
     All of the reactions produced a product peak after 1 hour incubation in HPLC analysis. Accordingly, the ligation method works for all combinations of nucleotides at the junctions to be joined. Optimisation to improve product yield is possible, but was not necessary as the results were conclusive and it was clear that the reaction was working for all combinations of nucleotides at the junctions to be joined. 
     Example 6 
     Effect of Different Modifications at the Ligation Site 
     6.1 Materials 
     Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) was produced as described in 5.1 and Chlorella virus DNA ligase (SEQ ID NO:29, commercially available as SplintR ligase, NEB) was purchased. 
     The following biotinylated template oligonucleotide and segment oligonucleotides (Table 9) were synthesized by standard solid phase methods. 
     
       
         
           
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Name 
                 Sequence (junction modification in bold) 
               
               
                   
               
             
            
               
                 Template 
                 5′-biotin TTTAGGTAAGCCGAGGTTTGGCC-3′ 
               
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
               
               
                 5′ segment (WT) 
                 5′-GGCCAAA-3′ 
               
               
                   
               
               
                 5′ segment (Mo1) 
                 5′-GGCCAA( OMe ) A -3′ 
               
               
                   
               
               
                 5′ segment (Mo2) 
                 5′-GGCCAA( F ) A -3′ 
               
               
                   
               
               
                 centre segment (WT) 
                 5′-(p)CCTCGG-3′ 
               
               
                   
               
               
                 centre segment (Mo4A) 
                 5′-(p)( O Me) C CTCGG-3′ 
               
               
                   
               
               
                 centre segment (Mo5A) 
                 5′-(p)( F ) C CTCGG-3′ 
               
               
                   
               
               
                 centre segment (Mo7) 
                 5′(p)( Me ) C CTCGG-3′ 
               
               
                   
               
               
                 3′ segment (WT) 
                 5′-(p)CTTACCT-3′ 
               
               
                   
               
               
                 3′ segment (Mo8) 
                 5′-(p)( Me )CTTACCT3′ 
               
               
                   
               
               
                 OMe indicates 2′ methoxy substitution on the ribose ring 
               
               
                 F indicates 2′ fluoro substitution on the ribose ring 
               
               
                 All remaining sugar residues are deoxyribose residues 
               
               
                 Me indicates 5-methyl cytosines 
               
            
           
         
       
     
     6.2 Method 
     Reactions were set up as follows: 
     
       
         
           
               
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                 Reaction 
                 5′ segment 
                 Centre segment 
                 3′ segment 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 WT 
                 WT 
                 WT 
               
               
                 2 
                 WT 
                 Mo4A 
                 WT 
               
               
                 3 
                 WT 
                 Mo5A 
                 WT 
               
               
                 4 
                 WT 
                 Mo7 
                 WT 
               
               
                 5 
                 Mo1 
                 WT 
                 WT 
               
               
                 6 
                 Mo1 
                 Mo4A 
                 WT 
               
               
                 7 
                 Mo1 
                 Mo5A 
                 WT 
               
               
                 8 
                 Mo1 
                 Mo7 
                 WT 
               
               
                 9 
                 Mo2 
                 WT 
                 WT 
               
               
                 10 
                 Mo2 
                 Mo4A 
                 WT 
               
               
                 11 
                 Mo2 
                 Mo5A 
                 WT 
               
               
                 12 
                 Mo2 
                 Mo7 
                 WT 
               
               
                 13 
                 WT 
                 WT 
                 Mo8 
               
               
                 14 
                 WT 
                 Mo4A 
                 Mo8 
               
               
                 15 
                 WT 
                 Mo5A 
                 Mo8 
               
               
                 16 
                 WT 
                 Mo7 
                 Mo8 
               
               
                 17 
                 Mo1 
                 WT 
                 Mo8 
               
               
                 18 
                 Mo1 
                 Mo4A 
                 Mo8 
               
               
                 19 
                 Mo1 
                 Mo5A 
                 Mo8 
               
               
                 20 
                 Mo1 
                 Mo7 
                 Mo8 
               
               
                 21 
                 Mo2 
                 WT 
                 Mo8 
               
               
                 22 
                 Mo2 
                 Mo4A 
                 Mo8 
               
               
                 23 
                 Mo2 
                 Mo5A 
                 Mo8 
               
               
                 24 
                 Mo2 
                 Mo7 
                 Mo8 
               
               
                   
               
            
           
         
       
     
     For each 50 μL reaction: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 3′-segment (1 mM, stock, 20 μM final) 
                 1 μl 
               
               
                   
                 centre segment (1 mM, stock, 20 μM final) 
                 1 μl 
               
               
                   
                 5′-segment (1 mM, stock, 20 μM final) 
                 1 μl 
               
               
                   
                 Template (1 mM, stock, 20 μM final) 
                 1 μl 
               
               
                   
                 H 2 O 
                 up to 50 μl 
               
               
                   
                   
               
            
           
         
       
     
     For Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 DNA ligase buffer (50 mM Tris-HCl, 1 mM DTT) 
                  5 μl 
               
               
                   
                 Mutant CC31 DNA ligase (0.45 mM) 
                 10 μl 
               
               
                   
                 MnCl 2  (50 mM) 
                  5 μl 
               
               
                   
                 ATP (10 mM) 
                 10 μl 
               
               
                   
                   
               
            
           
         
       
     
     Whereas for Chloralla virus DNA ligase (SEQ ID NO:29, commercially available as SplintR ligase, NEB) 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 NEB DNA ligase buffer (for Chlorella) 
                 5 μl 
               
               
                   
                 Chlorella virus DNA ligase 
                 2 μl 
               
               
                   
                   
               
            
           
         
       
     
     Each reaction mix was incubated at 20° C. 1 hour. Each reaction was terminated by heating at 95° C. for 10 minutes. HPLC analysis was carried out using the method of Example 1. 
     6.3 Results and Conclusions 
     All of the reactions produced a product peak in HPLC analysis. Accordingly, the ligation method works for all combinations of modifications tested at the junctions to be joined. Optimisation to improve product yield is possible, but was not necessary as the results were conclusive and it was clear that the reaction was working for all combinations of modifications tested at the junctions to be joined. 
     Example 7 
     Ability to Use Different Numbers of Segments to Build Larger Oligonucleotides 
     7.1 Materials 
     Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) was produced as described in 5.1. 
     The following biotinylated template DNA oligonucleotides and DNA segment oligonucleotides (Table 11) were synthesized by standard solid phase methods. 
     
       
         
           
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 Template 
                 5′-biotin 
               
               
                   
                 TTTGGTGCGAAGCAGAAGGTAAGCCGAGGT 
               
               
                   
                 TTGGCC-3′ (SEQ ID NO: 47) 
               
               
                   
               
               
                 5′ segment (1) 
                 5′-GGCCAAA-3′ 
               
               
                   
               
               
                 centre segment (2) 
                 5′-(p)CCTCGG-3′ 
               
               
                   
               
               
                 centre segment/ 
                 5′-(p)TCTGCT-3′ 
               
               
                 3′segment (5) 
                   
               
               
                   
               
               
                 centre segment (3) 
                 5′-(p)CTTACCT-3′ 
               
               
                   
               
               
                 3′ segment (4) 
                 5′-(p)TCGCACC-3′ 
               
               
                   
               
               
                 (p) = phosphate, 
               
            
           
         
       
     
     7.2 Method 
     Reactions were set up as follows: 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                 Centre 
                   
                 Total number 
               
               
                   
                 5′ segment 
                 segment(s) 
                 3′ segment 
                 of segments 
               
               
                   
                   
               
             
            
               
                   
                 1 
                 2 and 3 
                 5 
                 4 
               
               
                   
                 1 
                 2, 3 and 5 
                 4 
                 5 
               
               
                   
                   
               
            
           
         
       
     
     Reactions were run in phosphate buffered saline, pH=7.04 in a total volume of 100 μl and set up as follows: 
     Template (20 μM final) 
     Each segment (20 μM final) 
     MgCl 2  (10 mM final) 
     ATP (100 μM final) 
     Mutant CC31 DNA ligase (25 μM final) 
     Each reaction was incubated at 28° C. overnight before being terminated by heating at 94° C. for 1 minute. Products were analysed by HPLC mass spec. 
     7.3 Results and Conclusions 
     The reaction using 4 segments produced a fully ligated product of 27 base pairs in length. The reaction using 5 segments produced a product of 33 base pairs in length. In both cases the observed mass of the product was in concordance with that expected for the desired sequence. In conclusion, it is clearly possible to assemble multiple segments to generate oligonucleotides of the desired length and sequence as defined by the appropriate complementary template sequence. 
     Example 8 
     Assembly and Ligation of 5-10-5 Segments to Form a Gapmer, wherein the 5′ and the 3′ Segments Comprise (i) 2′-OMe Ribose Sugar Modifications, (ii) Phosphorothioate Linkages or (iii) 2′-OMe Ribose Sugar Modifications and Phosphorothioate Linkages; and wherein the Central Segment is Unmodified DNA 
     8.1 Materials 
     Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) was produced as described in 5.1 the following biotinylated template DNA oligonucleotide and segment oligonucleotides (Table 13) were synthesized by standard solid phase methods. 
     
       
         
           
               
               
             
               
                 TABLE 13 
               
               
                   
               
               
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 Template 
                 5′-biotin TTTGGTGCGAAGCAGACTGAGGC-3′ 
               
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 3′ segment (3OMe) 
                 5′-(p)(OMe)G(OMe)C(OMe)C(OMe)T(OMe)C-3′ 
               
               
                   
               
               
                 3′ segment 
                 5′-(p)(OMe)G*(0Me)C*(OMe)C*(OMe)T*(OMe)C-3′ 
               
               
                 (3PS + OMe) 
                   
               
               
                   
               
               
                 3′ segment (3PS) 
                 5′-(p)G*C*C*T*C-3′ 
               
               
                   
               
               
                 centre segment (D) 
                 5′-(p)AGTCTGCTTC-3′ 
               
               
                   
               
               
                 5′ segment (5OMe) 
                 5′-(OMe)G(OMe)C(OMe)A(OMe)C(OMe)C-3′ 
               
               
                   
               
               
                 5′ segment 
                 5′-(OMe)G*(OMe)C*(OMe)A*(OMe)C*(OMe)C-3′ 
               
               
                 (5PS + OMe) 
                   
               
               
                   
               
               
                 5′ segment (5PS) 
                 5′-G*C*A*C*C-3′ 
               
               
                   
               
               
                 OMe indicates 2′ methoxy substitution on the ribose ring 
               
               
                 All remaining sugar residues are deoxyribose residues 
               
               
                 *phosphorothioate 
               
            
           
         
       
     
     8.2 Method 
     Reactions were set up as follows: 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 14 
               
               
                   
                   
               
               
                   
                   
                   
                 Centre 
                   
               
               
                   
                 Reaction 
                 3′ segment 
                 segment(s) 
                 5′ segment 
               
               
                   
                   
               
             
            
               
                   
                 1 
                 3PS 
                 D 
                 5PS 
               
               
                   
                 2 
                 3OMe 
                 D 
                 5OMe 
               
               
                   
                 3 
                 3PS + OMe 
                 D 
                 5PS + OMe 
               
               
                   
                   
               
            
           
         
       
     
     Each of reactions 1, 2 and 3 were set up in 100 μl final volume in phosphate buffered saline with the following components: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 3′ segment 
                 20 μM final 
               
               
                   
                 Centre segment 
                 20 μM final 
               
               
                   
                 5′ segment 
                 20 μM final 
               
               
                   
                 Template 
                 20 μM final 
               
               
                   
                 MgCl 2   
                 10 mM final 
               
               
                   
                 ATP 
                 50 μM final 
               
               
                   
                 Enzyme 
                 25 μM final 
               
               
                   
                   
               
            
           
         
       
     
     Each reaction mix was incubated at 20° C. overnight. Each reaction was terminated by heating at 95° C. for 10 minutes. HPLC mass spec analysis was carried out. 
     8.3 Results and Conclusions 
     Product oligonucleotide corresponding to the successful ligation of all three fragments was produced in all three reactions. 
     Accordingly, mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) is able to ligate 3 segments together to form a ‘gapmer’ where the 5′ and 3′ ‘wings’ have a phosphorothioate backbone, whereas the central region has a phosphodiester backbone, and all the sugar residues in the gapmer are deoxyribose residues. Enterobacteria phage CC31 ligase (SEQ ID NO:23) is also able to ligate 3 segments together to form a ‘gapmer’ where the 5′ and 3′ ‘wings’ have 2′-methoxyribose (2′-OMe) residues, whereas the central region has deoxyribose residues, and all of the linkages are phosphodiester linkages. Finally, Enterobacteria phage CC31 ligase (SEQ ID NO:23) is able to ligate 3 segments together to form a ‘gapmer’ where the 5′ and 3′ ‘wings’ have the combined modifications (a phosphorothioate backbone and 2′-methoxyribose residues), whereas the central region has deoxyribose residues and phosphodiester linkages. 
     Example 9 
     Assembly and Ligation of Segments Comprising Locked Nucleic Acids (LNA) 
     9.1 Materials 
     Mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) was produced as described in 5.1. A mutant Staphylococcus aureus NAD dependent ligase (NAD-14) was produced as described in 13.1 
     The following biotinylated template DNA oligonucleotide and segment oligonucleotides (Table 15) were synthesized by standard solid phase methods. 
     
       
         
           
               
               
             
               
                 TABLE 15 
               
               
                   
               
               
                 Name 
                 Sequence 
               
               
                   
               
             
            
               
                 Template 
                 5′-biotin TTTGGTGCGAAGCAGACTGAGGC-3′ 
               
               
                   
                 (SEQ ID NO: 30) 
               
               
                   
               
               
                 5′-segment 
                 5′-GCCTCAG-3′ 
               
               
                   
               
               
                 LNA 5′-segment (oligo 1) 
                 5′-GCCTCA(LNA)G-3′ 
               
               
                   
               
               
                 Centre segment 
                 5′-(p)TCTGCT-3′ 
               
               
                   
               
               
                 LNA centre segment (oligo 
                 5′-(p)(LNA)TCTGCT-3′ 
               
               
                 2) 
               
               
                   
               
               
                 (p) = phosphate 
               
               
                 LNA = locked nucleic acid 
               
            
           
         
       
     
     9.2 Method 
     Reactions were set up as follows: 
     
       
         
           
               
             
               
                   
               
               
                 Reaction volume 100 μL 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 Template 
                 20 μM final 
               
               
                   
                 Enzyme 
                 25 μM final 
               
               
                   
                 All oligonucleotide segments 
                 20 μM final 
               
               
                   
                   
               
            
           
         
       
     
     Reactions were set up varying enzyme (mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) or NAD-14), divalent cation (Mg 2+  or Mn 2+ ) and combinations of oligonucleotide segments as set out in Table 16. 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 16 
               
               
                   
               
               
                   
                 SEQ ID 
                 SEQ ID 
                   
                   
               
               
                 Enzyme 
                 NO: 23 
                 NO: 23 
                 NAD-14 
                 NAD-14 
               
               
                   
               
             
            
               
                 Divalent 
                 10 mM 
                 10 mM 
                 10 mM 
                 10 mM 
               
               
                 cation 
                 MgCl 2   
                 MnCl 2   
                 MgCl 2   
                 MnCl 2   
               
               
                 Cofactor 
                 100 μM 
                 100 μM 
                 100 μM 
                 100 μM 
               
               
                   
                 ATP 
                 ATP 
                 NAD 
                 NAD 
               
               
                 Buffer 
                 PBS, 
                 PBS, 
                 50 mM 
                 50 mM 
               
               
                   
                 pH = 7.04 
                 pH = 7.04 
                 KH 2 PO 4 , 
                 KH 2 PO 4 , 
               
               
                   
                   
                   
                 pH 7.5 
                 pH 7.5 
               
               
                 5′ segment + 
                 Product 
                 Product 
                 Product 
                 Product 
               
               
                 Centre 
               
               
                 segment 
               
               
                 Oligo 1 + 
                 Product 
                 Product 
                 Product 
                 Product 
               
               
                 Centre 
               
               
                 segment 
               
               
                 5′ segment + 
                 Product 
                 Product 
                 Product 
                 Product 
               
               
                 Oligo 2 
               
               
                 Oligo 1 + 
                 No 
                 No 
                 No 
                 No 
               
               
                 oligo 2 
                 product 
                 product 
                 product 
                 product 
               
               
                   
               
            
           
         
       
     
     Each reaction mix was incubated at 28° C. overnight. Each reaction was terminated by heating at 94° C. for 1 minute. HPLC mass spec. analysis was carried out. 
     9.3 Results and Conclusions 
     Product oligonucleotide was produced in control reaction reactions (unmodified oligonucleotides only) and where a single locked nucleic acid was included in one segment at the ligation junction regardless of whether it was on the 3′ or 5′ side of the junction. When locked nucleic acids were included at both sides (oligo 1+oligo 2) no product was detected. The data was similar for both enzymes and regardless of whether Mg2+ or Mn2+ were used. 
     Enzyme mutations and/or selection screens could be carried out to identify an enzyme capable of ligating segments with a locked nucleic acid at both the 3′ and 5′ side of the junction. 
     Example 10 
     Assembly and Ligation of Three Segments (7-6-7) to Form a Gapmer wherein the 5′ and the 3′ Segments Comprise 2′ MOE Ribose Sugar Modifications and All Linkages are Phosphorothioate Linkages, Using a Variant of Enterobacteria Phage CC31 Ligase in the Presence of Mg 2+  or Mn 2+   
     10.1 Phosphorothioate Bond Formation 
     In order to determine whether a mutant Enterobacteria phage CC31 ligase (SEQ ID NO:23) was able to ligate modified oligonucleotide segments with a phosphorothioate backbone, 2′ MOE ribose sugar modifications and 5-methylated pyrimidine bases, reactions were performed using the oligonucleotide segments shown in table 15. Reactions were performed in the presence of Mg 2+  and Mn 2+  ions. 
     10.2 Materials 
     Oligonucleotides were chemically synthesised using standard methods as shown below: 
                         TABLE 17               Name   Sequence                  5′ segment 2′-   5′-mG*mG*mC*mC*mA*dA*dA-3′       MOE PS                   centre segment   5′-(p)*dC*dC*dT*dC*dG*dG -3′       PS                   3′ segment 2′-   5′-(p)*dC*dT*mU*mA*mC*mC*mU-3′       MOE PS                   Biotinylated   5′-biotin dT dT dT dA dG dG dT dA       template   dA dG dC dC dG dA dG dG dT dT dT           dG dG dC dC-3′ (SEQ ID NO: 2)               (p)* = 5′-phosphorothioate, * = phosphorothioate linkage, mX = MOE bases, dX = DNA bases       all segments and product have 5-methyl pyrimidines (with the exception of the template)       mT and m (Me)U are considered to be equivalent       N.B. the target 2′MOE PS molecule produced by ligation of the segments in table 17, when hybridised to the biotinylated template shown in table 17, is: 5′-mG*mG*mC*mC*mA*dA*dA*dC*dC*dT*dC*dG*dG*dC*dT*mU*mA*mC*mC*mU-3′ (SEQ ID NO: 1)       Purified mutant Enterobacteria phage CC31 ligase (SEQ ID NO: 23) was prepared as described in example 5.1. HPLC analysis was carried out.            
10.3 Oligonucleotide Assembly and Ligation Method with Enterobacteria Phage CC31 Ligase Variant (SEQ ID NO:23)
 
     Reactions were prepared as follows: 
     
       
         
           
               
             
               
                   
               
             
            
               
                 MgCl 2  reaction 
               
            
           
           
               
               
               
            
               
                 10 x T4 DNA ligase buffer (NEB)* 
                 5 
                 μl 
               
               
                 template 
                 20 
                 μM final concentration 
               
               
                 5′ segment 2′ MOE PS 
                 20 
                 μM final concentration 
               
               
                 centre segment PS 
                 20 
                 μM final concentration 
               
               
                 3′ segment 2′ MOE PS 
                 20 
                 μM final concentration 
               
               
                 ligase (24.3 μM) 
                 10 
                 μl 
               
            
           
           
               
               
            
               
                 water 
                 made up to 50 μL 
               
               
                   
               
            
           
           
               
            
               
                 *1 x buffer contains 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM ATP, 10 mM 
               
               
                 DTT, pH 7.5 
               
            
           
           
               
            
               
                 MnCl 2  reaction 
               
            
           
           
               
               
               
               
            
               
                   
                 10 x ligase buffer* 
                 5 
                 μl 
               
               
                   
                 ATP (10 mM) 
                 5 
                 μl 
               
               
                   
                 MnCl 2  (50 mM) 
                 5 
                 μl 
               
               
                   
                 template 
                 20 
                 μM final concentration 
               
               
                   
                 5′ segment 2′ MOE PS 
                 20 
                 μM final concentration 
               
               
                   
                 centre segment PS 
                 20 
                 μM final concentration 
               
               
                   
                 3′ segment 2′ MOE PS 
                 20 
                 μM final concentration 
               
               
                   
                 ligase (24.3 μM) 
                 10 
                 μl 
               
            
           
           
               
               
               
            
               
                   
                 water 
                 made up to 50 μL 
               
               
                   
                   
               
               
                   
                 *1 x buffer contains 50 mM Tris-HCl, 10 mM DTT, pH 7.5 
               
            
           
         
       
     
     The final reactions contained 20 μM of each segment and template, 5 mM MgCl 2  or 5 mM MnCl 2 , 1 mM ATP, 50 mM Tris-HCl, 10 mM DTT, pH 7.5 and 4.9 μM ligase. Additional reactions were prepared containing no enzyme and served as a negative control. Reactions were incubated for 16 hours at 25° C. and then quenched by heating to 95° C. for 5 minutes. Precipitated proteins were cleared by centrifugation and samples were analysed by HPLC. 
     10.4 Results and Conclusion 
     Product, template and segment oligonucleotides were clearly resolved in the control chromatogram and no ligation was observed. Ligase reactions performed in the presence of 5 mM MgCl 2  led to the formation of an intermediate product formed from the ligation of the 5′ segment and centre segments, but no full length product was detected. Ligase reactions performed in the presence of MnCl 2  produced both full length product and intermediate (5′ segment plus centre segment intermediate). Both ligase reactions showed that unligated oligonucleotide segments remained. However, optimisation of the protocol is possible in order to maximise product yield. 
     Example 11 
     Assembly and Ligation of Three Segments (7-6-7) to Form a Gapmer wherein the 5′ and the 3′ Segments Comprise 2′ MOE Ribose Sugar Modifications and All Linkages are Phosphorothioate Linkages, Using Wild-Type Chlorella Virus DNA Ligase in the Presence of Native Mg 2+   
     11.1 Materials 
     In order to determine whether Chlorella virus DNA ligase (SEQ ID NO:29, commercially available as SplintR ligase, NEB) was able to ligate modified oligonucleotide segments with a phosphorothioate backbone, 2′ MOE ribose sugar modifications and 5-methylated pyrimidine bases reactions were performed using the oligonucleotide segments shown in example 10.2 table 17. Reactions were performed at 25° C., 30° C. and 37° C. to investigate the effect of temperature on the enzyme activity. 
     11.2 Oligonucleotide Assembly and Ligation Method with Commercial Chlorella Virus DNA Ligase (SEQ ID NO:29) 
     Each Oligonucleotide segment and template were dissolved in nuclease free water as detailed below: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Biotinylated template 
                 249.6 ng/μl 
               
               
                   
                 5′ segment 2′ MOE PS 
                 182.0 ng/μl 
               
               
                   
                 Centre segment PS 
                 534.0 ng/μl 
               
               
                   
                 3′ segment 2′ MOE PS 
                 531.0 ng/μl 
               
               
                   
                   
               
            
           
         
       
     
     Reactions were prepared as follows: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 10 x buffer (NEB)* 
                 6 
                 μl 
               
               
                   
                 template 
                 3.8 
                 μl 
               
               
                   
                 5′ segment 2′ MOE PS 
                 18.1 
                 μl 
               
               
                   
                 centre segment PS 
                 4.8 
                 μl 
               
               
                   
                 3′ segment 2′ MOE PS 
                 6.3 
                 μl 
               
               
                   
                 water 
                 15 
                 μl 
               
               
                   
                 SplintR ligase (25 U/μl) 
                 6 
                 μl 
               
               
                   
                   
               
               
                   
                 *1 x buffer contains 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM ATP, 10 mM DTT, pH 7.5 
               
            
           
         
       
     
     The final reactions contained 20 μM of each segment and template, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM ATP, 10 mM DTT, pH 7.5 and 2.5 U/μl ligase. Reactions were incubated at 25° C., 30° C. and 37° C. Additional reactions were prepared containing no enzyme and served as a negative control. Following 16 hours incubation, reactions were quenched by heating to 95° C. for 10 minutes. Precipitated proteins were cleared by centrifugation and samples were analysed by HPLC. 
     11.3 Results and Conclusion 
     Product, template and segment oligonucleotides were clearly resolved in the control chromatogram and no ligation was observed. HPLC analysis of the ligase reactions showed that unligated oligonucleotide segments remained, but Chlorella virus DNA ligase was able to successfully ligate the segments. The activity of the ligase increased with increasing temperature. At 25° C. the Chlorella virus DNA ligase was able to successfully ligate the 5′ segment and centre segment but no full length product was observed. At 30° C. and 37° C., full length product was detected in addition to the intermediate formed from 5′ segment and centre segment. 
     Example 12 
     Screening a Panel of 15 ATP and NAD Ligases for Activity Towards the Ligation of Three Segments (7-6-7) to Form a Gapmer wherein the 5′ and the 3′ Segments Comprise 2′ MOE Ribose Sugar Modifications and All Linkages are Phosphorothioate Linkages 
     12.1 Materials 
     Wild-type ATP and NAD dependent ligases described in table 18 and 19 were each fused at the N-terminus to a CBD. Genes were synthesised, cloned into pET28a and expressed in  E. coli  BL21(DE3) using standard cloning, expression and extraction methods. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 ATP dependent Ligases 
               
            
           
           
               
               
               
            
               
                 Name 
                 Origin 
                 SEQ ID 
               
               
                   
               
               
                 M1I5D1_Pbcv 
                 
                   Paramecium bursaria 
                 
                 SEQ ID NO: 48 
               
               
                   
                 Chlorella virus NE-JV-4 
               
               
                 M1I998_Pbcv 
                 
                   Paramecium bursaria 
                 
                 SEQ ID NO: 49 
               
               
                   
                 Chlorella virus NYs1 
               
               
                 M1HX09_Pbcv 
                 
                   Paramecium bursaria 
                 
                 SEQ ID NO: 50 
               
               
                   
                 Chlorella virus NE-JV-1 
               
               
                 M1HUL0_Atcv 
                 
                   Acanthocystis turfacea 
                 
                 SEQ ID NO: 51 
               
               
                   
                 Chlorella virus Canal-1 
               
               
                 M1HRK1_Atcv 
                 
                   Acanthocystis turfacea 
                 
                 SEQ ID NO: 52 
               
               
                   
                 Chlorella virus Br0604L 
               
               
                 M1I273_Atcv 
                 
                   Acanthocystis turfacea 
                 
                 SEQ ID NO: 53 
               
               
                   
                 Chlorella virus NE-JV-2 
               
               
                 M1I600_Atcv 
                 
                   Acanthocystis turfacea 
                 
                 SEQ ID NO: 54 
               
               
                   
                 Chlorella virus TN603.4.2 
               
               
                 M1H4A4_Atcv 
                 
                   Acanthocystis turfacea 
                 
                 SEQ ID NO: 55 
               
               
                   
                 Chlorella virus GM0701.1 
               
               
                 F5B464_Sphage 
                 
                   Synechococcus 
                 
                 SEQ ID NO: 56 
               
               
                   
                 phage S-CRM01 
               
               
                 A0A0F9M1S3 
                 marine sediment metagenome - 
                 SEQ ID NO: 57 
               
               
                   
                 uncharacterized protein 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 NAD dependent ligases 
               
            
           
           
               
               
               
            
               
                 Name 
                 Origin 
                 SEQ ID 
               
               
                   
               
               
                 MtNAD 
                 
                   Mycobacterium tuberculosis 
                 
                 SEQ ID NO: 58 
               
               
                   
                 (strain ATCC 25618/H37RV) 
               
               
                 EfNAD 
                 
                   Enterococcus faecalis 
                 
                 SEQ ID NO: 59 
               
               
                   
                 (strain ATCC 700802/V583) 
               
               
                 HiNAD 
                 
                   Haemophilus influenzae 
                 
                 SEQ ID NO: 60 
               
               
                   
                 (strain ATCC 51907/DSM 11121/KW20/Rd) 
               
               
                 SaNAD 
                 
                   Staphylococcus aureus 
                 
                 SEQ ID NO: 61 
               
               
                 SpNAD 
                 
                   Streptococcus pneumoniae 
                 
                 SEQ ID NO: 62 
               
               
                   
                 (strain P1031) 
               
               
                   
               
            
           
         
       
     
     CBD-Ligase fusions were bound to PERLOZA beads as described in 1.4 with the following modifications. CBD-ligase fusion proteins were grown from a single colony of BL21(DE3) cells (NEB) and grown in a 50 mL expression culture. The cells were harvested by centrifugation, resuspended in 5-10 mL Tris-HCl (50 mM, pH 7.5) and lysed by sonication. The lysate was cleared by centrifugation and 1 mL of PERLOZA 100 (PERLOZA) beads (50% slurry, pre-equilibrated with 50 mM Tris-HCl pH 7.5) was added to the supernatant which was shaken at 20° C. for 1 hour. The PERLOZA cellulose beads were then collected and washed with 30 ml buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Tween 20, 10% Glycerol) followed by 10 ml Tris-HCl (50 mM, pH 7.5) and were finally re-suspended in 1 mL Tris-HCl (50 mM, pH 7.5). In order to analyse protein expression, 20 μl of the PERLOZA bead slurry was mixed with 20 μl of SDS loading buffer and incubated at 95° C. for 5 minutes before being run on a SDS PAGE gradient gel (4-20%) according to a standard protocol. 
     12.2 2′ MOE and Phosphorothioate Modified Oligonucleotide Assembly and Ligation Method 
     Modified oligonucleotide segments with a phosphorothioate backbone, 2′ MOE ribose sugar modifications and 5-methylated pyrimidine bases shown in example 10.2 table 17 were used. Each oligonucleotide segment and template was dissolved in nuclease free water as detailed below: 
     
       
         
           
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Biotinylated template 
                 1500 ng/μl 
               
               
                   
                 5′ segment 2′ MOE PS 
                 1008 ng/μl 
               
               
                   
                 Centre segment PS 
                  725 ng/μl 
               
               
                   
                 3′ segment 2′ MOE PS 
                 1112 ng/μl 
               
               
                   
                   
               
            
           
         
       
     
     ATP Assay mix was prepared as follows: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 template 
                 85.6 
                 μl 
               
               
                   
                 5′ segment 2′ MOE PS 
                 43.5 
                 μl 
               
               
                   
                 centre segment PS 
                 46.8 
                 μl 
               
               
                   
                 3′ segment 2′ MOE PS 
                 40.1 
                 μl 
               
               
                   
                 DTT 1M 
                 8 
                 μl 
               
               
                   
                 MgCL2 1M 
                 4 
                 μl 
               
               
                   
                 ATP (50 mM) 
                 16 
                 μl 
               
               
                   
                 Tris 0.5M 
                 80 
                 μl 
               
               
                   
                 water 
                 476 
                 μl 
               
               
                   
                   
               
            
           
         
       
     
     NAD Assay mix was prepared as follows: 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 template 
                 85.6 
                 μl 
               
               
                   
                 5′ segment 2′ MOE PS 
                 43.5 
                 μl 
               
               
                   
                 centre segment PS 
                 46.8 
                 μl 
               
               
                   
                 3′ segment 2′ MOE PS 
                 40.1 
                 μl 
               
               
                   
                 DTT 1M 
                 8 
                 μl 
               
               
                   
                 MgCL2 1M 
                 4 
                 μl 
               
               
                   
                 NAD (50 mM) 
                 1.6 
                 μl 
               
               
                   
                 Tris 0.5M 
                 80 
                 μl 
               
               
                   
                 water 
                 490.4 
                 μl 
               
               
                   
                   
               
            
           
         
       
     
     Each immobilized protein (40 μl, 50% PERLOZA bead slurry) was pipetted into a PCR tube. The beads were pelleted by centrifugation and the supernatant was removed by pipetting. Assay mix (40 μl) was added to each reaction (The final reactions contained 20 μM of each segment and template, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM ATP or 100 μM NAD, 10 mM DTT, pH 7.5, and 40 μl of ligase on PERLOZA beads). A reaction containing no protein served as a negative control. Reactions were incubated for 18 hours at 30° C. and then quenched by heating to 95° C. for 10 minutes. Precipitated proteins were cleared by centrifugation and samples were analysed by HPLC. 
     12.3 Results and Conclusion 
     Product, template and segment oligonucleotides were clearly resolved in the control chromatogram and no ligation was observed. HPLC analysis of the ligase reactions showed that all proteins catalyzed the successful ligation of the 5′ segment and centre segment to form an intermediate product, but only some ligases catalyzed the ligation of all three segments to yield the full length product as described in table 20. The NAD dependent ligase from  Staphylococcus aureus  (SaNAD, SEQ ID NO:61) yielded the most full length product. Optimisation to improve product yield is possible and within the skilled person&#39;s skill set. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 20 
               
             
            
               
                   
                   
               
               
                   
                 SED ID 
                 Conversion (%)* 
               
            
           
           
               
               
               
               
            
               
                 Gene name 
                 NO 
                 intermediate 
                 product 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 M1I5D1_Pbcv 
                 48 
                 5.0 
                 0.8 
               
               
                 M1I998_Pbcv 
                 49 
                 5.1 
                 0.0 
               
               
                 M1HX09_Pbcv 
                 50 
                 14.4 
                 6.6 
               
               
                 M1HUL0_Atcv 
                 51 
                 9.5 
                 2.0 
               
               
                 M1HRK1_Atcv 
                 52 
                 2.8 
                 9.4 
               
               
                 M1I273_Atcv 
                 53 
                 12.0 
                 6.4 
               
               
                 M1I600_Atcv 
                 54 
                 8.8 
                 5.1 
               
               
                 M1H4A4_Atcv 
                 55 
                 2.1 
                 0.0 
               
               
                 F5B464_Sphage 
                 56 
                 2.1 
                 0.0 
               
               
                 A0A0F9M1S3_ms_metagenome 
                 57 
                 1.4 
                 0.0 
               
               
                 MtNAD 
                 58 
                 7.1 
                 0.0 
               
               
                 EfNAD 
                 59 
                 19.1 
                 0.0 
               
               
                 HiNAD 
                 60 
                 0.3 
                 0.0 
               
               
                 SaNAD 
                 61 
                 10.5 
                 11.8 
               
               
                 SpNAD 
                 62 
                 19.0 
                 0.0 
               
               
                   
               
               
                 *Conversion was calculated from the HPLC peak area relative to the template which is not consumed in the reaction and serves as an internal standard. Conversion = product area/(template + product area)*100 
               
            
           
         
       
     
     Example 13 
     Semi-Continuous Ligation Reaction 
     13.1 Materials 
     A mutant  Staphylococcus aureus  ligase (NAD-14) fused at the N-terminal to a CBD was produced using standard cloning, expression and extraction methods. CBD-NAD-14 mutant ligase was then bound to PERLOZA beads: 50 ml of protein lysate was added to 7.5 ml PERLOZA beads, incubated at room temperature for 1 hour and then the beads were collected in a glass column (BioRad Econo-Column 10 cm length, 2.5 cm diameter #7372512). The beads were washed with 200 ml Buffer Y (50 mM Tris8, 500 mM NaCl, 0.1% Tween 20, 10% Glycerol), then with 200 ml Buffer Z (50 mM Tris8, 200 mM NaCl, 0.1% Tween 20, 10% Glycerol) and 200 ml PBS. The estimated concentration of mutant NAD-14 ligase on the beads was 69 μM of ligase per ml of beads. 
     The following template DNA oligonucleotide and segment oligonucleotides (Table 21) were synthesized by standard solid phase methods. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 21 
               
               
                   
               
               
                   
                   
                   
                 % HPLC 
               
               
                 Segment 
                 Sequence 
                 MW 
                 purity 
               
               
                   
               
             
            
               
                 Template 3 
                 5′ TTTGGTGCGAAGCAGACTGAGGC-3′ 
                   
                   
               
               
                   
                 (SEQ ID NO: 30) 
                   
                   
               
               
                   
               
               
                 centre segment 
                 5′-(p)dTdCdTdGdCdT-3′ 
                 1865.4 
                 97.5 
               
               
                   
               
               
                 MOE 3′- 
                 5′-(p)dTdCmGmCmAmCmC-3′ 
                 2546.7 
                 98.0 
               
               
                 segment 
                   
                   
                   
               
               
                   
               
               
                 MOE 5′- 
                 5′-mGmCmCmUmCdAdG-3′ 
                 2492.7 
                 98.3 
               
               
                 segment 
               
               
                   
               
               
                 (p)= phosphate, mX = MOE bases, dX = DNA bases 
               
               
                 all 5-methyl pyrimidines 
               
               
                 all linkages are phosphodiester linkages 
               
            
           
         
       
     
     A “tri-template hub” (approximately 24 kDa) comprising a support material referred to as the “hub” and three template sequences was produced ( FIG. 11 ). Each copy of the template was covalently attached, at its own individual attachment point, to the “hub”. The “tri-template hub” molecule is a higher molecular weight than the target (product) oligonucleotide (100% complementary to the template sequence), thereby allowing it to be retained when the impurities and products are separated from the reaction mixture. It should be noted that in this particular case the template sequence was SEQ ID NO:30 and three copies were attached to the hub. In the following example, a tri-template hub is also produced, but with a different template sequence. Accordingly, as the template sequence varies between examples, so too does the tri-template hub. 
     The following reaction mix (total volume 5 ml) was prepared: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
             
            
               
                 250 
                 μl 
                 1M KH 2 PO 4 , pH 7.5 
                 (50 
                 mM final) 
               
               
                 108 
                 μl 
                 0.07011M centre segment 
                 (1.5 
                 mM final) 
               
               
                 137 
                 μl 
                 0.05481M 3′-segment 
                 (1.5 
                 mM final) 
               
               
                 168 
                 μl 
                 0.04461M 5′-segment 
                 (1.5 
                 mM final) 
               
               
                 750 
                 μl 
                 0.00387M Hub (Template) 
                 (0.55 
                 mM final) 
               
               
                 350 
                 μl 
                 50 mM NAD +   
                 (3.5 
                 mM final) 
               
               
                 1000 
                 μl 
                 50 mM MgCl 2   
                 (10 
                 mM final) 
               
               
                 2237 
                 μl 
                 Nuclease free H 2 O 
               
               
                   
               
            
           
         
       
     
     13.2 Methods 
     A semi-continuous system was set up as shown in  FIG. 12 . 
     4 ml of PERLOZA beads and immobilised mutant NAD-14 ligase were packed into a Pharmacia XK16 column (B). A water bath and peristaltic pump (C) was used to keep the temperature of the column, using the column water compartment, at 30° C. The beads were equilibrated by running 120 ml (30× column volume) of buffer containing 50 mM KH 2 PO 4  at pH7.5 for 120 minutes at 1 ml/min. An AKTA explorer pump A1 (A) was used to create the flow through the Pharmacia XK16 column. 
     Following column equilibration, the 5 ml reaction mix (mixed well by vortexing) was loaded onto the column, collected in the reservoir tube (D) and recirculated through the column using the AKTA explorer A1 pump. The reaction mix was recirculated through the system at a flow rate of 1 ml/min in continuous circulation mode for 16 hours. Samples were collected after 30 minutes, 60 minutes, 90 minutes, 4 hours, 5 hours, 6 hours, 7 hours, 14 hours and 16 hours for HPLC analysis. 
     13.3 Results and Conclusions 
       
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 22 
               
               
                   
               
               
                   
                   
                 Centre 
                   
                 5′ + centre 
                   
               
               
                   
                 5′-segment 
                 segment 
                 3′-segment 
                 intermediate 
                 Product 
               
               
                 Sample 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
                 (%) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 30 
                 min 
                 7.80 
                 11.00 
                 24.00 
                 39.40 
                 4.40 
               
               
                 60 
                 min 
                 3.30 
                 4.50 
                 18.80 
                 41.50 
                 18.90 
               
               
                 90 
                 min 
                 2.10 
                 2.80 
                 15.10 
                 33.80 
                 34.40 
               
               
                 4 
                 hr 
                 1.80 
                 2.40 
                 9.60 
                 19.20 
                 56.80 
               
               
                 5 
                 hr 
                 1.72 
                 2.40 
                 8.30 
                 15.70 
                 61.50 
               
               
                 6 
                 hr 
                 1.69 
                 2.50 
                 8.60 
                 15.90 
                 69.40 
               
               
                 7 
                 hr 
                 1.70 
                 2.40 
                 8.30 
                 14.80 
                 70.90 
               
               
                 14 
                 hr 
                 2.00 
                 2.70 
                 1.60 
                 5.40 
                 88.10 
               
               
                 16 
                 hr 
                 0.80 
                 1.90 
                 1.70 
                 3.30 
                 89.50 
               
               
                   
               
            
           
         
       
     
     The percentage of each segment, intermediate and product is expressed as fractional peak area relative to the tri-template hub peak area. 
     In conclusion, the semi-continuous flow reaction worked and after 16 hours the reaction was almost complete. 
     Example 14 
     Separating Oligonucleotides of Different Sizes by Filtration: a) Separation of a 20-mer Oligonucleotide (SEQ ID NO:1) and a Hub Comprising Three Non-Complementary 20-mer Oligonucleotides (SEQ ID NO:30); (b) Separation of a 20-mer Oligonucleotide (SEQ ID NO:1) from Segment 6-mer and 8-mer Oligonucleotides (See Table 1) and a Hub Comprising Three Complementary 20-mer Oligonucleotides (SEQ ID NO:2) 
     14.1 Materials 
     All oligonucleotides used were synthesized by standard solid phase methods. 
     A tri-template hub, as described in 13.1 ( FIG. 11 ), was used. 
     A variety of filters of varying molecular weight cut-offs and from different manufacturers were used as shown in Tables 23 and 24. 
     14.2 Methods 
     14.2.1 Dead-End Filtration Set-Up and Protocol for Screening of Polymeric Membranes: (Protocol 1) 
     A dead-end filtration rig was set-up as shown in  FIG. 13  comprising a MET dead-end filtration cell placed in a water bath sitting on a magnetic stirrer and hotplate. Pressure inside the cell was provided from a flow of nitrogen. 
     The coupon of membrane (14 cm 2 ) to be tested was first cut to the appropriate size and placed in the cell. The membrane was first conditioned with HPLC grade water (200 ml) and then with PBS buffer (200 ml). The cell was then depressurised, the remaining PBS solution was removed and replaced by a solution containing oligonucleotides (40 ml of oligonucleotides in PBS at a 1 g/L concentration). The cell was placed on a hot stirrer plate and the solution was heated to the desired temperature while being stirred using magnetic agitation. Pressure was applied to the cell (aiming for approximately 3.0 bar; the actual pressure was recorded in each case). Stirring of the solution was either stopped or continued and permeate solution was collected (approximately 20 ml) and analysed by HPLC. Flux was recorded. The system was then depressurised to allow sampling and analysis by HPLC of the retentate solution. More PBS buffer (20 ml) was then added to the filtration cell and the previous procedure was repeated 3 times. The membrane was finally washed with PBS buffer. 
     All samples were analysed by HPLC without any dilution. 
     14.2.2 Cross-Flow Filtration Set-Up and Protocol for Screening of Polymeric Membranes: (Protocol 2) 
     A cross-flow filtration rig was set-up as shown in  FIG. 14 . The feed vessel ( 1 ) consisting of a conical flask contained the oligonucleotide solution to be purified. The solution was pumped to a cross-flow filtration cell ( 4 ) using an HPLC pump ( 2 ) while temperature within the cell was maintained using a hot plate ( 3 ). The solution within the cell was recirculated using a gear pump ( 6 ). A pressure gauge ( 5 ) enabled the pressure to be read during the experiment. Samples of the retentate solution were taken from the sampling valve ( 7 ) while the permeate solution was sampled from the permeate collection vessel ( 8 ). 
     The coupon of membrane to be tested was first cut to the appropriate size and placed in the cell. The system was washed with a PBS solution (100 ml). Temperature of the solution was adjusted to the desired set point. A solution containing oligonucleotide products in PBS (7.5 ml at 1 g/L) was fed into the system. PBS solution was then pumped into the system using the HPLC pump at a flow rate matching the flow rate of the permeate solution (typically 3 ml/min). Pressure was recorded using the pressure gauge. The retentate solution was sampled for HPLC analysis every 5 diafiltration volumes. The permeate solution was sampled for HPLC analysis every diafiltration volume. The experiment was stopped after 20 diafiltration volumes. 
     In the case of the experiment using the Snyder membrane having a 5 kDa molecular weight cut off (lot number 120915R2) and SEQ ID NO:46 and SEQ ID NO:30 the above methodology was modified as follows. The coupon of membrane to be tested was first cut to the appropriate size and placed in the cell. The system was washed with a Potassium phosphate solution (100 ml, 50 mM, pH 7.5). Temperature of the solution was adjusted to the desired set point. A solution containing oligonucleotide products in potassium phosphate (approximately 1 g/L) was added to ethylenediaminetetraacetic acid (EDTA) (230 μL of a 500 mM solution). The solution was then fed into the system. Potassium phosphate buffer was then pumped into the system using the HPLC pump at a flow rate matching the flow rate of the permeate solution (typically 4 ml/min). Pressure was recorded using the pressure gauge. The retentate solution was sampled for HPLC analysis every 5 diafiltration volumes. The permeate solution was sampled for HPLC analysis every diafiltration volume. The experiment was stopped after 15 diafiltration volumes 
     14.3 Results 
       
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 results for the dead-end filtration experiments following protocol 1 (14.2.1) 
               
            
           
           
               
               
            
               
                   
                 Rejection (%) 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Stirring 
                   
                 MWCO 
                 Lot 
                 SEQ 
                 Temp. 
                   
                 Tri-template 
               
               
                 (Yes or No) 
                 Membrane 
                 (kDa) 
                 number 
                 ID NO 
                 (° C.) 
                 Product 
                 hub 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Yes 
                 NADIR 
                 10 
                 226162 
                 1 and 30 
                 60 
                 38 
                 96 
               
               
                 Yes 
                 NADIR 
                 10 
                 226162 
                 1 and 2 
                 60 
                 90 
                 98 
               
               
                   
                   
                   
                   
                   
                 80 
                 40 
                 43 
               
               
                 Yes 
                 NADIR 
                 10 
                 226162 
                 1 and 2 
                 60 
                 83 
                 92 
               
               
                   
                   
                   
                   
                   
                 65 
                 77 
                 84 
               
               
                   
                   
                   
                   
                   
                 70 
                 38 
                 41 
               
               
                   
                   
                   
                   
                   
                 75 
                 12 
                 20 
               
               
                 Yes 
                 NADIR 
                 5 
                 226825 
                 1 and 2 
                 60 
                 97 
                 95 
               
               
                   
                   
                   
                   
                   
                 65 
                 99 
                 93 
               
               
                   
                   
                   
                   
                   
                 70 
                 97 
                 92 
               
               
                   
                   
                   
                   
                   
                 75 
                 90 
                 95 
               
               
                 No 
                 NADIR 
                 5 
                 226825 
                 1 and 2 
                 75 
                 66 
                 96 
               
               
                 No 
                 NADIR 
                 10 
                 226162 
                 1 and 2 
                 75 
                 80 
                 80 
               
               
                 No 
                 Snyder 
                 5 
                 120915R2 
                 1 and 2 
                 75 
                 97 
                 98 
               
               
                 No 
                 Osmonics 
                 5 
                 622806PT 
                 1 and 2 
                 75 
                 99 
                 98 
               
               
                 No 
                 Osmonics 
                 10 
                 622806PW 
                 1 and 2 
                 75 
                 86 
                 95 
               
               
                   
               
               
                 MWCO = molecular weight cut-off 
               
            
           
         
       
     
     In the experiment using the 10 kDa MWCO NADIR membrane at 60° C., clear separation between the product sequence (SEQ ID NO:1) and the non-complementary tri-template hub (comprising SEQ ID NO:30) was demonstrated.  FIG. 15A  shows a chromatogram of the retentate solution, which remained in the filtration cell and contained mainly tri-template hub, after two diafiltration volumes; and  FIG. 15B  a chromatogram of the permeate, solution enriched in the product, after two diafiltration volumes. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 results for the cross-flow filtration experiments (following protocol 2) (14.2.2) 
               
            
           
           
               
               
               
               
            
               
                   
                 Diafil- 
                 Rejection (%) 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 MWCO 
                 Lot 
                 SEQ 
                 Temp. 
                   
                 tration 
                 Seg- 
                 Seg- 
                 Seg- 
                   
                 Tri-template 
                 
                   
                 
               
               
                 Membrane 
                 (kDa) 
                 number 
                 ID NO 
                 (° C.) 
                 Pressure 
                 volume 
                 ment 1 
                 ment 2 
                 ment 3 
                 Product 
                 hub 
                 
                   
                 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Osmonics 
                 10 
                 622806PW 
                 1 and 2 
                 75 
                 3.1 
                 0 
                 N/A 
                 N/A 
                 N/A 
                 100 
                 88 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 5 
                 N/A 
                 N/A 
                 N/A 
                 64 
                 88 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 10 
                 N/A 
                 N/A 
                 N/A 
                 40 
                 82 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 15 
                 N/A 
                 N/A 
                 N/A 
                 12 
                 74 
                 
                   
                 
               
               
                 Snyder 
                 5 
                 120915R2 
                 1 and 2 
                 75 
                 3.1 
                 0 
                 N/A 
                 N/A 
                 N/A 
                 100 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                 82 
                   
                 5 
                 N/A 
                 N/A 
                 N/A 
                 88 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                 75 
                   
                 10 
                 N/A 
                 N/A 
                 N/A 
                 98 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                 75 
                   
                 15 
                 N/A 
                 N/A 
                 N/A 
                 98 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                 75 
                   
                 20 
                 N/A 
                 N/A 
                 N/A 
                 96 
                 100 
                 
                   
                 
               
               
                 Snyder 
                 5 
                 120915R2 
                 1 and 2 
                 85 
                 3.1 
                 0 
                 N/A 
                 N/A 
                 N/A 
                 99 
                 99 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 5 
                 N/A 
                 N/A 
                 N/A 
                 63 
                 99 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 10 
                 N/A 
                 N/A 
                 N/A 
                 79 
                 99 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 15 
                 N/A 
                 N/A 
                 N/A 
                 100 
                 98 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 20 
                 N/A 
                 N/A 
                 N/A 
                 100 
                 99 
                 
                   
                 
               
               
                 Snyder 
                 5 
                 120915R2 
                 1 and 2 + 
                 50 
                 3.0 
                 0 
                 100  
                 100  
                 100  
                 100 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                 segments 
                   
                   
                 5 
                 39 
                 39 
                 40 
                 100 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                 from table 1 
                   
                   
                 10 
                 82 
                 86 
                 86 
                 100 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 15 
                 77 
                 81 
                 79 
                 100 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 20 
                 * 
                 * 
                 * 
                 100 
                 100 
                 
                   
                 
               
               
                 Snyder 
                 5 
                 120915R2 
                 1 and 2 
                 80 
                 3.1 
                 0 
                 N/A 
                 N/A 
                 N/A 
                 96 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 5 
                 N/A 
                 N/A 
                 N/A 
                 22 
                 96 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 10 
                 N/A 
                 N/A 
                 N/A 
                 10 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 20 
                 N/A 
                 N/A 
                 N/A 
                 38 
                 100 
                 
                   
                 
               
               
                 Snyder 
                 5 
                 120915R2 
                 46 and 30 
                 80 
                 3.4 
                 0 
                 N/A 
                 N/A 
                 N/A 
                 98 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 5 
                 N/A 
                 N/A 
                 N/A 
                 83 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 10 
                 N/A 
                 N/A 
                 N/A 
                 68 
                 100 
                 
                   
                 
               
               
                   
                   
                   
                   
                   
                   
                 15 
                 N/A 
                 N/A 
                 N/A 
                 72 
                 100 
                 
                   
                 
               
               
                   
               
               
                 MWCO = molecular weight cut-off 
               
               
                 * concentration of solutes too low preventing meaningful analysis 
               
               
                     indicates data missing or illegible when filed 
               
            
           
         
       
     
     In the experiment using the 5 kDa MWCO Snyder membrane at 50° C. and 3.0 bar pressure, clear separation between the segment sequences (see table 1) and the complementary tri-template hub (comprising SEQ ID NO:2) and product (SEQ ID NO:1) was demonstrated.  FIG. 16A  shows a chromatogram of the retentate solution, which contained mainly tri-template hub and product, after 20 diafiltration volumes; and  FIG. 16B  a chromatogram of the permeate, which contained mainly segment oligonucleotides, after 20 diafiltration volumes. 
     In the experiment using the 5 kDa MWCO Snyder membrane at 80° C. and 3.1 bar pressure, clear separation between the complementary tri-template hub (comprising SEQ ID NO:2) and product (SEQ ID NO:1) was demonstrated.  FIG. 17A  shows a chromatogram of the retentate solution, which contained tri-template hub only, after 20 diafiltration volumes; and  FIG. 17B  a chromatogram of the permeate solution, which contained the product only, after 2 diafiltration volumes. 
     14.4 Conclusions 
     Oligonucleotides of different lengths and molecular weights can be separated using filtration. As shown above, the type of membrane and the conditions, such as temperature, affect the level of separation. For a given set of oligonucleotides of different lengths/molecular weights, suitable membranes and conditions can be selected to allow the required separation. For example, we have demonstrated that segment oligonucleotides (shortmers of 6 and 8 nucleotides in length), as outlined in table 1, can be separated from the product oligonucleotide (20-mer oligonucleotide having SEQ ID NO:1) and tri-template hub (comprising 3×20-mer of SEQ ID NO:2 attached to a solid support), and the product oligonucleotide and tri-template hub can, in turn, be separated from each other. 
     Overall Conclusions 
     We have shown that it is possible to synthesize oligonucleotides, including oligonucleotides with a range of therapeutically relevant chemical modifications, in solution by assembling short oligonucleotide segments on a complementary template, ligating the segments together and separating the product oligonucleotide from both impurities and its complementary template in an efficient process that is scalable and suitable for large scale therapeutic oligonucleotide manufacture. 
     By synthesizing oligonucleotides in solution we have avoided the scale up constraints imposed by solid phase methods. In using the inherent properties of DNA to recognise complementary sequences specifically and bind complementary sequences with an affinity that reflects both the fidelity of the complementary sequence and the length of the complementary sequence we have been able to produce oligonucleotides of high purity without the need for chromatography. This both improves the efficiency of the production process and the scalability of the process. By recovering the template in an unchanged state during the separation process we are able to reuse the template for further rounds of synthesis and so have avoided the economic consequences of having to make one equivalent of template for every equivalent of product oligonucleotide formed. 
     Finally, although wild type ligases are known to ligate normal DNA effectively, we have shown that modifications to DNA result in decreased ligation efficiency and multiple modifications to the DNA are additive in their effect on decreasing the efficiency of ligation which can, in some cases render the DNA ligase completely ineffective. We have shown that by appropriate mutation and evolution of DNA ligases, ligation efficiency can be restored and appropriately modified DNA ligases are effective catalysts for synthesizing oligonucleotides which contain multiple modifications. 
     
       
         
           
               
             
               
                   
               
               
                 SEQUENCE LISTING 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 SEQ ID NO 
                 Sequence Identifier 
               
               
                   
               
               
                  1 
                 Example 1 desired product oligonucleotide sequence (″target″) 
               
               
                   
               
               
                  2 
                 Example 1-4,6, 10 and 11 template oligonucleotide sequence 
               
               
                   
               
               
                  3 
                 Wild type T4 DNA ligase protein sequence 
               
               
                   
               
               
                  4 
                 Wild type T4 DNA ligase protein sequence (when fused to CBD) 
               
               
                   
               
               
                  5 
                 Example 2 target sequence 
               
               
                   
               
               
                  6 
                 Wild-type Enterobacteria phage CC31 DNA ligase protein sequence 
               
               
                   
               
               
                  7 
                 Wild-type Enterobacteria phage CC31 DNA ligase protein sequence (when 
               
               
                   
                 fused to CBD) 
               
               
                   
               
               
                  8 
                 Wild-type Shigella phage Shf125875 DNA ligase protein sequence 
               
               
                   
               
               
                  9 
                 Wild-type Shigella phage Shf125875 DNA ligase protein sequence (when 
               
               
                   
                 fused to CBD) 
               
               
                   
               
               
                 10 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 11 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 12 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 13 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 14 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 15 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 16 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 17 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 18 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 19 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 20 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 21 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 22 
                 Mutant ligase (T4 backbone) protein sequence 
               
               
                   
               
               
                 23 
                 Mutant ligase (Enterobacteria phage CC31 backbone-clone A4) protein 
               
               
                   
                 sequence 
               
               
                   
               
               
                 24 
                 Mutant ligase (Enterobacteria phage CC31 backbone) protein sequence 
               
               
                   
               
               
                 25 
                 Mutant ligase (Enterobacteria phage CC31 backbone) protein sequence 
               
               
                   
               
               
                 26 
                 Mutant ligase (Enterobacteria phage CC31 backbone) protein sequence 
               
               
                   
               
               
                 27 
                 Mutant ligase (Enterobacteria phage CC31 backbone) protein sequence 
               
               
                   
               
               
                 28 
                 Mutant ligase ( Shigella  phage Shf125875 backbone) protein sequence 
               
               
                   
               
               
                 29 
                 Wild-type  Chlorella  ligase protein sequence 
               
               
                   
               
               
                 30 
                 Example 5, 8 and 9 template oligonucleotide sequence 
               
               
                   
               
               
                 31 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 32 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 33 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 34 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 35 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 36 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 37 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 38 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 39 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 40 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 41 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 42 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 43 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 44 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 45 
                 Example 5 template oligonucleotide sequence 
               
               
                   
               
               
                 46 
                 Example 14 ″20 mer″ oligonucleotide sequence 
               
               
                   
               
               
                 47 
                 Example 7 template oligonucleotide sequence 
               
               
                   
               
               
                 48 
                   Paramecium   bursaria   Chlorella  virus NE-JV-4 ligase 
               
               
                   
               
               
                 49 
                   Paramecium bursaria   Chlorella  virus NYs1 ligase 
               
               
                   
               
               
                 50 
                   Paramecium   bursaria   Chlorella  virus NE-JV-1 ligase 
               
               
                   
               
               
                 51 
                   Acanthocystis   turfacea   Chlorella  virus Canal-1 ligase 
               
               
                   
               
               
                 52 
                   Acanthocystis   turfacea   Chlorella  virus Br0604L ligase 
               
               
                   
               
               
                 53 
                   Acanthocystis   turfacea   Chlorella  virus NE-JV-2 ligase 
               
               
                   
               
               
                 54 
                   Acanthocystis   turfacea   Chlorella  virus TN603.4.2 ligase 
               
               
                   
               
               
                 55 
                   Acanthocystis   turfacea   Chlorella  virus GM0701.1 ligase 
               
               
                   
               
               
                 56 
                   Synechococcus  phage S-CRM01 ligase 
               
               
                   
               
               
                 57 
                 marine sediment metagenome ligase 
               
               
                   
               
               
                 58 
                   Mycobacterium   tuberculosis  (strain ATCC 25618/H37Rv) ligase 
               
               
                   
               
               
                 59 
                   Enterococcus   faecalis  (strain ATCC 700802N583) ligase 
               
               
                   
               
               
                 60 
                   Haemophilus  influenzae (strain ATCC 51907/DSM 11121/ KW20/Rd) ligase 
               
               
                   
               
               
                 61 
                   Staphylococcus   aureus  ligase 
               
               
                   
               
               
                 62 
                   Streptococcus   pneumoniae  (strain P1031) ligase 
               
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 1 
               
               
                 GGCCAAACCTCGGCTTACCT 
               
               
                   
               
               
                 SEQ ID NO: 2 
               
               
                 TTTAGGTAAGCCGAGGTTTGGCC 
               
               
                   
               
               
                 SEQ ID NO: 3 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 4 
               
               
                 GSILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIDVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 5 
               
               
                 CCC CAA ACC UCG GCU UAC Cu 
               
               
                   
               
               
                 SEQ ID NO: 6 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKEVITIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 7 
               
               
                 GSILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDML 
               
               
                 YLLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQP 
               
               
                 QMLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIE 
               
               
                 MTKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSL 
               
               
                 KGTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHN 
               
               
                 LDEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKEVITIDLRIVDIYEHSKQPGKAGGFYL 
               
               
                 ESECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLF 
               
               
                 LPIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 8 
               
               
                 MILDILNQIAAIGSTKTKQEILKKNKDNKLLERVYRLTYARGIQYYIKKWPGPGERSQAYGLLELD 
               
               
                 DMLDFIEFTLATRKLTGNAAIKELMGYIADGKPDDVEVLRRVMMRDLEVGASVSIANKVWPGLI 
               
               
                 QLQPQMLASAYDEKLITKNIKWPAFAQLKADGARCFAEVRDDGVQFFSRAGNEYHGLTLLADE 
               
               
                 LMEMTKEARERHPNGVLIDGELVYHSFDIKKAVSSGNDLSFLFGDNEESEEVQVADRSTSNGL 
               
               
                 ANKSLQGTISPKEAEGMVLQAWDYVPLDEVYSDGKIKGQKYDVRFAALENMAEGFKRIEPIEN 
               
               
                 QLVHNLDEAKVVYKKYVDQGLEGIILKNRDSYWENKRSKNLIKFKEVIDIALEVVGYYEHSKDPN 
               
               
                 KLGGVELVSRCRRITTDCGSGFKDTTHKTVDGVKVLIPLDERHDLDRERLMAEAREGKLIGRIA 
               
               
                 DCECNGWVHSKGREGTVGIFLPIIKGFRFDKTEADSFEDVFGPWSQTGL 
               
               
                   
               
               
                 SEQ ID NO: 9 
               
               
                 GSILDILNQIAAIGSTKTKQEILKKNKDNKLLERVYRLTYARGIQYYIKKWPGPGERSQAYGLLEL 
               
               
                 DDMLDFIEFTLATRKLTGNAAIKELMGYIADGKPDDVEVLRRVMMRDLEVGASVSIANKVWPGL 
               
               
                 IQLQPQMLASAYDEKLITKNIKWPAFAQLKADGARCFAEVRDDGVQFFSRAGNEYHGLTLLADE 
               
               
                 LMEMTKEARERHPNGVLIDGELVYHSFDIKKAVSSGNDLSFLFGDNEESEEVQVADRSTSNGL 
               
               
                 ANKSLQGTISPKEAEGMVLQAWDYVPLDEVYSDGKIKGQKYDVRFAALENMAEGFKRIEPIEN 
               
               
                 QLVHNLDEAKVVYKKYVDQGLEGIILKNRDSYWENKRSKNLIKFKEVIDIALEVVGYYEHSKDPN 
               
               
                 KLGGVELVSRCRRITTDCGSGFKDTTHKTVDGVKVLIPLDERHDLDRERLMAEAREGKLIGRIA 
               
               
                 DCECNGWVHSKGREGTVGIFLPIIKGFRFDKTEADSFEDVFGPWSQTGL 
               
               
                   
               
               
                 SEQ ID NO: 10 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKRVIDVDLKIVGIYPHRKDPTKA 
               
               
                 GGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDY 
               
               
                 VKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 11 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKGVIDVDLKIVGIYPHRKDPTKA 
               
               
                 GGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDY 
               
               
                 VKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 12 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKKVIDVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 13 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVILVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 14 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIKVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 15 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIQVDLKIVGIYPHRKDPTKA 
               
               
                 GGFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDY 
               
               
                 VKLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 16 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIVVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 17 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEVIRVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 18 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEAIDVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 19 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKEKIDVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 20 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKRVIVVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 21 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKKVIEVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 22 
               
               
                 MILKILNEIASIGSTKQKQAILEKNKDNELLKRVYRLTYSRGLQYYIKKWPKPGIATQSFGMLTLT 
               
               
                 DMLDFIEFTLATRKLTGNAAIEELTGYITDGKKDDVEVLRRVMMRDLECGASVSIANKVWPGLIP 
               
               
                 EQPQMLASSYDEKGINKNIKFPAFAQLKADGARCFAEVRGDELDDVRLLSRAGNEYLGLDLLK 
               
               
                 EELIKMTAEARQIHPEGVLIDGELVYHEQVKKEPEGLDFLFDAYPENSKAKEFAEVAESRTASN 
               
               
                 GIANKSLKGTISEKEAQCMKFQVWDYVPLVEIYSLPAFRLKYDVRFSKLEQMTSGYDKVILIENQ 
               
               
                 VVNNLDEAKVIYKKYIDQGLEGIILKNIDGLWENARSKNLYKFKKVIHVDLKIVGIYPHRKDPTKAG 
               
               
                 GFILESECGKIKVNAGSGLKDKAGVKSHELDRTRIMENQNYYIGKILECECNGWLKSDGRTDYV 
               
               
                 KLFLPIAIRLREDKTKANTFEDVFGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 23 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKRVIVIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 24 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKKVIKIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 25 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKGVIFIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 26 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKGVILIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 27 
               
               
                 MILDIINEIASIGSTKEKEAIIRRHKDNELLKRVFRMTYDGKLQYYIKKWDTRPKGDIHLTLEDMLY 
               
               
                 LLEEKLAKRVVTGNAAKEKLEIALSQTSDADAEVVKKVLLRDLRCGASRSIANKVWKNLIPEQPQ 
               
               
                 MLASSYDEKGIEKNIKFPAFAQLKADGARAFAEVRGDELDDVKILSRAGNEYLGLDLLKQQLIEM 
               
               
                 TKEARERHPGGVMIDGELVYHASTLPAGPLDDIFGDLPELSKAKEFKEESRTMSNGLANKSLK 
               
               
                 GTISAKEAAGMKFQVWDYVPLDVVYSEGKQSGFAYDVRFRALELMVQGYSQMILIENHIVHNL 
               
               
                 DEAKVIYRKYVDEGLEGIILKNIGAFWENTRSKNLYKFKRVIFIDLRIVDIYEHSKQPGKAGGFYLE 
               
               
                 SECGLIKVKAGSGLKDKPGKDAHELDRTRIWENKNDYIGGVLESECNGWLAAEGRTDYVKLFL 
               
               
                 PIAIKMRRDKDVANTFADIWGDFHEVTGL 
               
               
                   
               
               
                 SEQ ID NO: 28 
               
               
                 MILDILNQIAAIGSTKTKQEILKKNKDNKLLERVYRLTYARGIQYYIKKWPGPGERSQAYGLLELD 
               
               
                 DMLDFIEFTLATRKLTGNAAIKELMGYIADGKPDDVEVLRRVMMRDLEVGASVSIANKVWPGLI 
               
               
                 QLQPQMLASAYDEKLITKNIKWPAFAQLKADGARCFAEVRDDGVQFFSRAGNEYHGLTLLADE 
               
               
                 LMEMTKEARERHPNGVLIDGELVYHSFDIKKAVSSGNDLSFLFGDNEESEEVQVADRSTSNGL 
               
               
                 ANKSLQGTISPKEAEGMVLQAWDYVPLDEVYSDGKIKGQKYDVRFAALENMAEGFKRIEPIEN 
               
               
                 QLVHNLDEAKVVYKKYVDQGLEGIILKNRDSYWENKRSKNLIKFKRVIVIALEWGYYEHSKDPN 
               
               
                 KLGGVELVSRCRRITTDCGSGFKDTTHKTVDGVKVLIPLDERHDLDRERLMAEAREGKLIGRIA 
               
               
                 DCECNGWVHSKGREGTVGIFLPIIKGFRFDKTEADSFEDVFGPWSQTGL 
               
               
                   
               
               
                 SEQ ID NO: 29 
               
               
                 MAITKPLLAATLENIEDVQFPCLATPKIDGIRSVKQTQMLSRTFKPIRNSVMNRLLTELLPEGSDG 
               
               
                 EISIEGATFQDTTSAVMTGHKMYNAKFSYYWFDYVTDDPLKKYIDRVEDMKNYITVHPHILEHA 
               
               
                 QVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMKQFKDAEATII 
               
               
                 SMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSIEVDYDGVVFSIGTGFDADQRRDFW 
               
               
                 QNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR 
               
               
                   
               
               
                 SEQ ID NO: 30 
               
               
                 TTTGGTGCGAAGCAGACTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 31 
               
               
                 TTTGGTGCGAAGCAGAGTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 32 
               
               
                 TTTGGTGCGAAGCAGATTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 33 
               
               
                 TTTGGTGCGAAGCAGAATGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 34 
               
               
                 TTTGGTGCGAAGCAGTCTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 35 
               
               
                 TTTGGTGCGAAGCAGTGTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 36 
               
               
                 TTTGGTGCGAAGCAGTTTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 37 
               
               
                 TTTGGTGCGAAGCAGTATGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 38 
               
               
                 TTTGGTGCGAAGCAGCCTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 39 
               
               
                 TTTGGTGCGAAGCAGCGTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 40 
               
               
                 TTTGGTGCGAAGCAGCTTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 41 
               
               
                 TTTGGTGCGAAGCAGCATGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 42 
               
               
                 TTTGGTGCGAAGCAGGCTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 43 
               
               
                 TTTGGTGCGAAGCAGGGTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 44 
               
               
                 TTTGGTGCGAAGCAGGTTGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 45 
               
               
                 TTTGGTGCGAAGCAGGATGAGGC 
               
               
                   
               
               
                 SEQ ID NO: 46 
               
               
                 GCCUCAGTCTGCTTCGCACC 
               
               
                   
               
               
                 SEQ ID NO: 47 
               
               
                 TTTGGTGCGAAGCAGAAGGTAAGCCGAGGTTTGGCC 
               
               
                   
               
               
                 SEQ ID NO: 48 
               
               
                 MAITKPLLAATLENIEDVQFPCLATPKIDGIRSVKQTQMLSRTFKPIRNSVMNRLLTELLPEGSDG 
               
               
                 EISIEGATFQDTTSAVMTGHKMYNAKFSYYWFDYVTDDPLKKYSDRVEDMKNYITAHPHILDHE 
               
               
                 QVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTLKEGILLKMKQFKDAEATII 
               
               
                 SMTALFKNTNTKTKDNFGYSKRSTHKNGKVEEDVMGSIEVDYDGVVFSIGTGFDADQRRDFW 
               
               
                 QNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDH 
               
               
                   
               
               
                 SEQ ID NO: 49 
               
               
                 MTIAKPLLAATLENLDDVKFPCLVTPKIDCIRSLKQQHMLSRTFKPIRNSVMNKLLSELLPEGAD 
               
               
                 GEICIEDSTFQATTSAVMTGHKVYDEKFSYYWFDYWDDPLKSYTDRVNDMKKYVDDHPHILE 
               
               
                 HEQVKIIPLIPVEINNIDELSQYERDVLAKGFEGVMIRRPDGKYKFGRSTLKEGILLKMKQFKDAE 
               
               
                 ATIISMSPRLKNTNAKSKDNLGYSKRSTHKSGKVEEETMGSIEVDYDGWFSIGTGFDDEQRKH 
               
               
                 FWENKDSYIGKLLKFKYFEMGSKDAPRFPVFIGIRHEEDC 
               
               
                   
               
               
                 SEQ ID NO: 50 
               
               
                 MTAIQKPLLAASFKKLTVADVKYPVFATPKLDGIRALKIDGAFVSRTFKPIRNRAIADALQDLLPN 
               
               
                 GSDGEILSGSTFQDASSAVMTAKAGIGANTIFYWFDYVKDDPNKPYLDRMTDMENYLKERPEIL 
               
               
                 NDDRIKIVPLIPKKIETKDELDTFEKICLDQGFEGVMIRSGAGKYKFGRSTEKEGILIKIKQFEDDE 
               
               
                 AVVIGFTPMQTNTNDKSMNELGDMKRSSHKDGKVNLDTLGALEVDWNGITFSIGTGFDHALRD 
               
               
                 KLWSERDKLIGKIVKFKYFAQGVKTAPRFPVFIGFRDPDDM 
               
               
                   
               
               
                 SEQ ID NO: 51 
               
               
                 MAIQKPLLAASLKKMSVGDLTFPVFATPKLDGIRALKVGGTIVSRTFKPVRNSAISEVLASILPDG 
               
               
                 SDGEILSGKTFQESTSTVMTADAGLGSGTMFFWFDYVKDDPNKGYLDRIADMKSFTDRHPEIL 
               
               
                 KDKRVTIVPLFPKKIDTTEELHEFEKWCLDQGFEGVMVRNAGGKYKFGRSTEKEQILVKIKQFE 
               
               
                 DDEAVVIGVSALQTNTNDKKLNQLGEMRRTSHQDGKVELEMLGALDVDWNGIRFSIGTGFDRD 
               
               
                 TRVDLWKRREGVIGKIVKFKYFSQGIKTAPRFPVFLGFRDKDDM 
               
               
                   
               
               
                 SEQ ID NO: 52 
               
               
                 MAIQKPLLAASLKKLSVDDLTFPVYATPKLDGIRALKIDGTLVSRTFKPIRNTTISKVLTSLLPDGS 
               
               
                 DGEILSGKTFQDSTSTVMSADAGIGSGTTFFWFDYVKDDPNKGYLDRIADIKKFIDCRPEILKDS 
               
               
                 RVIIVPLFPKKIDTAEELNVFEKWCLDQGFEGVMVRNAGGKYKFGRSTEKEQILVKIKQFEDDEA 
               
               
                 VVIGVSALQTNTNDKKVNELGEMRRTSHQDGKVDLDMLGALDVDWNGIRFCIGTGFDKDTRE 
               
               
                 DLWKRRDSIIGKIVKFKYFSQGVKTAPRFPVFLGFRDKNDM 
               
               
                   
               
               
                 SEQ ID NO: 53 
               
               
                 MAIQKPLLAASLKKLSVDDLTFPVYATPKLDGIRALKIDGTIVSRTFKPIRNTTISNVLMSLLPDGS 
               
               
                 DGEILSGKTFQDSTSTVMSADAGIGSGTTFFWFDYVKDDPDKGYLDRIADMKKFVDSHPEILKD 
               
               
                 RRVTIVPLIPKKIDTVEELNVFEQWCLDQGFEGVMVRNAGGKYKFGRSTEKEQILVKIKQFEDD 
               
               
                 EAWIGVSALQTNVNDKKMNELGDMRRTSHKDGKIDLEMLGALDVEWNGIRFGIGTGFDKDTR 
               
               
                 EDLWKKRDSIIGKWKFKYFSQGIKTAPRFPVFLGFRDENDM 
               
               
                   
               
               
                 SEQ ID NO: 54 
               
               
                 MAIQKPLLAASLKKMSVDNLTFPVYATPKLDGIRALKIDGTLVSRTFKPIRNTTISKVLASLLPDGS 
               
               
                 DGEILSGKTFQDSTSTVMTTDAGIGSDTTFFWFDYVKDDPDKGYLDRIADMKTFVDQHPEILKD 
               
               
                 SCVTIVPLFPKKIDTPEELHVFEKWCLDQGFEGVMVRTAGGKYKFGRSTEKEQILVKIKQFEDD 
               
               
                 EAWIGVSALQTNTNDKKLNQLGEMRRTSHQDGKVDLDMLGALDVDWNGIRFSIGTGFDKDTR 
               
               
                 EDLWKQRDSIVGKVVKFKYFSQGIKTAPRFPVFLGFRDENDM 
               
               
                   
               
               
                 SEQ ID NO: 55 
               
               
                 MAIQKPLLAASLKKMSVDDLTFPVYTTPKLDGIRALKIDGTLVSRTFKPVRNSAISEVLASLLPDG 
               
               
                 SDGEILSGKTFQDSTSTVMTTDAGIGSDTTFFWFDYVKDDPNKGYLDRIADMKTFIDQHPEMLK 
               
               
                 DNHVTIVPLIPKKIDTVEELNIFEKWCLDQGFEGVMVRNAGGKYKFGRSTEKEQILVKIKQFEDD 
               
               
                 EAWIGVSALQTNTNDKKLNQLGEMRRTSHQDGKIDLEMLGALDVDWNGIRFSIGTGFDRDTR 
               
               
                 VDLWKRRDGIVGRTIKFKYFGQGIKTAPRFPVFLGFRDKDDM 
               
               
                   
               
               
                 SEQ ID NO: 56 
               
               
                 MLAGNFDPKKAKFPYCATPKIDGIRFLMVNGRALSRTFKPIRNEYIQKLLSKHLPDGIDGELTCG 
               
               
                 DTFQSSTSAIMRIAGEPDFKAWIFDYVDPDSTSILPFIERFDQISDIIYNGPIPFKHQVLGQSILYNI 
               
               
                 DDLNRYEEACLNEGYEGVMLRDPYGTYKFGRSSTNEGILLKVKRFEDAEATVIRIDEKMSNQNI 
               
               
                 AEKDNFGRTKRSSCLDGMVPMETTGALFVRNSDGLEFSIGSGLNDEMRDEIWKNKSSYIGKLV 
               
               
                 KYKYFPQGVKDLPRHPVFLGFRDPDDM 
               
               
                   
               
               
                 SEQ ID NO: 57 
               
               
                 MDAHELMKLNEYAERQNQKQKKQITKPMLAASLKDITQLDYSKGYLATQKLDGIRALMIDGKLV 
               
               
                 SRTFKPIRNNHIREMLEDVLPDGADGEIVCPGAFQATSSGVMSANGEPEFIYYMFDYVKDDITK 
               
               
                 EYWRRTQDMVQWLINQGPTRTPGLSKLKLLVPTLIKNYDHLKTYETECIDKGFEGVILRTPDSP 
               
               
                 YKCGRSTAKQEWLLKLKRFADDEAWIGFTEKMHNDNEATKDKFGHTVRSSHKENKRPAGTL 
               
               
                 GSLIVRDIKTEIEFEIGTGFDDELRQKIWDARPEWDGLCVKYKHFAISGVKEKPRFPSFIGVRDV 
               
               
                 EDM 
               
               
                   
               
               
                 SEQ ID NO: 58 
               
               
                 MSSPDADQTAPEVLRQWQALAEEVREHQFRYYVRDAPIISDAEFDELLRRLEALEEQHPELRT 
               
               
                 PDSPTQLVGGAGFATDFEPVDHLERMLSLDNAFTADELAAWAGRIHAEVGDAAHYLCELKIDG 
               
               
                 VALSLVYREGRLTRASTRGDGRTGEDVTLNARTIADVPERLTPGDDYPVPEVLEVRGEVFFRL 
               
               
                 DDFQALNASLVEEGKAPFANPRNSAAGSLRQKDPAVTARRRLRMICHGLGHVEGFRPATLHQ 
               
               
                 AYLALRAWGLPVSEHTTLATDLAGVRERIDYWGEHRHEVDHEIDGVVVKVDEVALQRRLGSTS 
               
               
                 RAPRWAIAYKYPPEEAQTKLLDIRVNVGRTGRITPFAFMTPVKVAGSTVGQATLHNASEIKRKG 
               
               
                 VLIGDTVVIRKAGDVIPEVLGPVVELRDGSEREFIMPTTCPECGSPLAPEKEGDADIRCPNARGC 
               
               
                 PGQLRERVFHVASRNGLDIEVLGYEAGVALLQAKVIADEGELFALTERDLLRTDLFRTKAGELS 
               
               
                 ANGKRLLVNLDKAKAAPLWRVLVALSIRHVGPTAARALATEFGSLDAIAAASTDQLAAVEGVGP 
               
               
                 TIAAAVTEWFAVDWHREIVDKWRAAGVRMVDERDESVPRTLAGLTIVVTGSLTGFSRDDAKEAI 
               
               
                 VARGGKAAGSVSKKTNYVVAGDSPGSKYDKAVELGVPILDEDGFRRLLADGPASRT 
               
               
                   
               
               
                 SEQ ID NO: 59 
               
               
                 MEQQPLTLTAATTRAQELRKQLNQYSHEYYVKDQPSVEDYVYDRLYKELVDIETEFPDLITPDS 
               
               
                 PTQRVGGKVLSGFEKAPHDIPMYSLNDGFSKEDIFAFDERVRKAIGKPVAYCCELKIDGLAISLR 
               
               
                 YENGVFVRGATRGDGTVGENITENLRTVRSVPMRLTEPISVEVRGECYMPKQSFVALNEEREE 
               
               
                 NGQDIFANPRNAAAGSLRQLDTKIVAKRNLNTFLYTVADFGPMKAKTQFEALEELSAIGFRTNP 
               
               
                 ERQLCQSIDEVWAYIEEYHEKRSTLPYEIDGIVIKVNEFALQDELGFTVKAPRWAIAYKFPPEEA 
               
               
                 ETVVEDIEVVTIGRTGVVTPTAVMAPVRVAGTTVSRASLHNADFIQMKDIRLNDHVIIYKAGDIIPE 
               
               
                 VAQVLVEKRAADSQPYEMPTHCPICHSELVHLDEEVALRCINPKCPAQIKEGLNHFVSRNAMNI 
               
               
                 DGLGPRVLAQMYDKGLVKDVADLYFLTEEQLMTLDKIKEKSANNIYTAIQGSKENSVERLIFGLG 
               
               
                 IRHVGAKAAKILAEHFGDLPTLSRATAEEIVALDSIGETIADSVVTYFENEEVHELMAELEKAQVN 
               
               
                 LTYKGLRTEQLAEVESPFKDKTVVLTGKLAQYTREEAKEKIENLGGKVTGSVSKKTDIVVAGED 
               
               
                 AGSKLTKAESLGVTVWNEQEMVDALDASHF 
               
               
                   
               
               
                 SEQ ID NO: 60 
               
               
                 MTNIQTQLDNLRKTLRQYEYEYHVLDNPSVPDSEYDRLFHQLKALELEHPEFLTSDSPTQRVG 
               
               
                 AKPLSGFSQIRHEIPMLSLDNAFSDAEFNAFVKRIEDRLILLPKPLTFCCEPKLDGLAVSILYVNG 
               
               
                 ELTQAATRGDGTTGEDITANIRTIRNVPLQLLTDNPPARLEVRGEVFMPHAGFERLNKYALEHN 
               
               
                 EKTFANPRNAAAGSLRQLDPNITSKRPLVLNAYGIGIAEGVDLPTTHYARLQWLKSIGIPVNPEIR 
               
               
                 LCNGADEVLGFYRDIQNKRSSLGYDIDGTVLKINDIALQNELGFISKAPRWAIAYKFPAQEELTLL 
               
               
                 NDVEFQVGRTGAITPVAKLEPVFVAGVTVSNATLHNGDEIERLNIAIGDTVVIRRAGDVIPQIIGVL 
               
               
                 HERRPDNAKPIIFPTNCPVCDSQIIRIEGEAVARCTGGLFCAAQRKEALKHFVSRKAMDIDGVG 
               
               
                 GKLIEQLVDRELIHTPADLFKLDLTTLTRLERMGAKSAENALNSLENAKSTTLARFIFALGIREVG 
               
               
                 EATALNLANHFKTLDALKDANLEELQQVPDVGEWANRIFIFWREAHNVAVVEDLIAQGVHWET 
               
               
                 VEVKEASENLFKDKTVVLTGTLTQMGRNEAKALLQQLGAKVSGSVSSKTDFVIAGDAAGSKLA 
               
               
                 KAQELNITVLTEEEFLAQITR 
               
               
                   
               
               
                 SEQ ID NO: 61 
               
               
                 MADLSSRVNELHDLLNQYSYEYYVEDNPSVPDSEYDKLLHELIKIEEEHPEYKTVDSPTVRVGG 
               
               
                 EAQASFNKVNHDTPMLSLGNAFNEDDLRKFDQRIREQIGNVEYMCELKIDGLAVSLKYVDGYF 
               
               
                 VQGLTRGDGTTGEDITENLKTIHAIPLKMKEPLNVEVRGEAYMPRRSFLRLNEEKEKNDEQLFA 
               
               
                 NPRNAAAGSLRQLDSKLTAKRKLSVFIYSVNDFTDFNARSQSEALDELDKLGFTTNKNRARVN 
               
               
                 NIDGVLEYIEKWTSQRESLPYDIDGIVIKVNDLDQQDEMGFTQKSPRWAIAYKFPAEEVVTKLLD 
               
               
                 IELSIGRTGVVTPTAILEPVKVAGTTVSRASLHNEDLIHDRDIRIGDSVVVKKAGDIIPEVVRSIPER 
               
               
                 RPEDAVTYHMPTHCPSCGHELVRIEGEVALRCINPKCQAQLVEGLIHFVSRQAMNIDGLGTKIIQ 
               
               
                 QLYQSELIKDVADIFYLTEEDLLPLDRMGQKKVDNLLAAIQQAKDNSLENLLFGLGIRHLGVKAS 
               
               
                 QVLAEKYETIDRLLTVTEAELVEIHDIGDKVAQSVVTYLENEDIRALIQKLKDKHVNMIYKGIKTSD 
               
               
                 IEGHPEFSGKTIVLTGKLHQMTRNEASKWLASQGAKVTSSVTKNTDVVIAGEDAGSKLTKAQSL 
               
               
                 GIEIVVTEQQFVDKQNELNS 
               
               
                   
               
               
                 SEQ ID NO: 62 
               
               
                 MNKRMNELVALLNRYATEYYTSDNPSVSDSEYDRLYRELVELETAYPEQVLADSPTHRVGGKV 
               
               
                 LDGFEKYSHQYPLYSLQDAFSREELDAFDARVRKEVAHPTYICELKIDGLSISLTYEKGILVAGVT 
               
               
                 RGDGSIGENITENLKRVKDIPLTLPEELDITVRGECYMPRASFDQVNQARQENGEPEFANPRNA 
               
               
                 AAGTLRQLDTAVVAKRNLATFLYQEASPSTRDSQEKGLKYLEQLGFVVNPKRILAENIDEIWNFI 
               
               
                 QEVGQERENLPYDIDGVVIKVNDLASQEELGFTVKAPKWAVAYKFPAEEKEAQLLSVDWTVGR 
               
               
                 TGVVTPTANLTPVQLAGTTVSRATLHNVDYIAEKDIRKDDTVIVYKAGDIIPAVLRVVESKRVSEE 
               
               
                 KLDIPTNCPSCNSDLLHFEDEVALRCINPRCPAQIMEGLIHFASRDAMNITGLGPSIVEKLFAANL 
               
               
                 VKDVADIYRLQEEDFLLLEGVKEKSAAKLYQAIQASKENSAEKLLFGLGIRHVGSKASQLLLQYF 
               
               
                 HSIENLYQADSEEVASIESLGGVIAKSLQTYFATEGSEILLRELKETGVNLDYKGQTVVADAALS 
               
               
                 GLTVVLTGKLERLKRSEAKSKLESLGAKVTGSVSKKTDLVVVGADAGSKLQKAQELGIQVRDEA 
               
               
                 WLESL