# Patent

## Title
Methods and apparatus for synthesizing nucleic acid

## Abstract

The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and specially designed nucleotide analogs. Using the methods of the invention, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Because the nucleotide analogs have an unmodified 3′ OH, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in natural polynucleotides suitable for incorporation into biological systems.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Non-provisional patent application Ser. No. 14/459,014, filed Aug. 13, 2014, which is a continuation-in-part of U.S. Non-provisional patent application Ser. No. 14/056,687, filed Oct. 17, 2013, now issued as U.S. Pat. No. 8,808,989, which claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 61/891,162, filed Oct. 15, 2014 and U.S. Provisional Patent Application Ser. No. 61/807,327, filed Apr. 2, 2013. This application additionally claims priority to U.S. Provisional Patent Application Ser. No. 62/069,067, filed Oct. 27, 2014 and U.S. Provisional Patent Application Ser. No. 62/038,604, filed Aug. 18, 2014. The contents of each of the above applications are incorporated by reference herein in their entireties.


FIELD OF THE INVENTION
The invention relates to methods and apparatus for synthesizing polynucleotides (de novo) with a desired sequence and without the need for a template. As such, the invention provides the capacity to make libraries of polynucleotides of varying sequence and varying length for research, genetic engineering, and gene therapy.
BACKGROUND
Genetic engineering requires tools for determining the content of genetic material as well as tools for constructing desired genetic materials. The tools for determining the content of genetic material have made it possible to sequence an entire human genome in about one day for under $1,000. (See Life Technologies, Press Release: Benchtop Ion ProtonTM Sequencer, Jan. 10, 2012). In contrast, the tools for constructing desired genetic materials, e.g., de novo DNA synthesis, have not improved at the same pace. As a point of reference, over the past 25 years, the cost (per base) of de novo small nucleic acid synthesis has dropped 10-fold, while the cost (per base) of nucleic acid sequencing has dropped over 10,000,000-fold. The lack of progress in DNA synthesis now limits the pace of translational genomics, i.e., whereby the role of individual sequence variations are determined and used to develop therapeutic treatments.
Currently, most de novo nucleic acid sequences are synthesized using solid phase phosphoramidite-techniques developed more than 30 years ago. The technique involves the sequential de-protection and synthesis of sequences built from phosphoramidite reagents corresponding to natural (or non-natural) nucleic acid bases. Phosphoramidite nucleic acid synthesis is length-limited, however, in that nucleic acids greater than 200 base pairs (bp) in length experience high rates of breakage and side reactions. Additionally, phosphoramidite synthesis produces toxic by-products, and the disposal of this waste limits the availability of nucleic acid synthesizers, and increases the costs of contract oligo production. (It is estimated that the annual demand for oligonucleotide synthesis is responsible for greater than 300,000 gallons of hazardous chemical waste, including acetonitrile, trichloroacetic acid, toluene, tetrahydrofuran, and pyridine. See LeProust et al., Nucleic Acids Res., vol. 38(8), p. 2522-2540, (2010), incorporated by reference herein in its entirety). Thus, there is a need for more efficient and cost-effective methods for oligonucleotide synthesis.
SUMMARY
The invention provides improved methods for nucleic acid synthesis. Methods of the invention provide faster and longer de novo synthesis of polynucleotides. As such, the invention dramatically reduces the overall cost of synthesizing custom nucleic acids. Methods of the invention are directed to template-independent synthesis of polynucleotides by using a nucleotidyl transferase enzyme to incorporate nucleotide analogs having an unmodified 3′ hydroxyl coupled to an inhibitor by a cleavable linker. Because of the inhibitor, synthesis pauses with the addition of each new base, whereupon the linker is cleaved, separating the inhibitor and leaving a polynucleotide that is essentially identical to a naturally occurring nucleotide (i.e., is recognized by the enzyme as a substrate for further nucleotide incorporation).
The invention additionally includes an apparatus that utilizes methods of the invention for the production of custom polynucleotides. An apparatus of the invention includes one or more bioreactors providing aqueous conditions and a plurality of sources of nucleotide analogs. The bioreactor may be e.g., a reservoir, a flow cell, or a multi-well plate. Starting from a solid support, the polynucleotides are grown in the reactor by adding successive nucleotides via the natural activity of a nucleotidyl transferase, e.g., a terminal deoxynucleotidyl transferase (TdT) or any other enzyme which elongates DNA or RNA strands without template direction. Upon cleavage of the linker, a natural polynucleotide is exposed on the solid support. Once the sequence is complete, the support is cleaved away, leaving a polynucleotide essentially equivalent to that found in nature. In some embodiments, the apparatus is designed to recycle nucleotide analog solutions by recovering the solutions after nucleotide addition and reusing solutions for subsequence nucleotide addition. Thus, less waste is produced, and the overall cost per base is reduced as compared to state-of-the-art methods. In certain embodiments, a bioreactor may include a microfluidic device and/or use inkjet printing technology.
Terminating groups may include, for example, charged moieties or steric inhibitors. In general, large macromolecule that prevent nucleotidyl transferase enzymes from achieving a functional conformation can be used to inhibit oligonucleotide synthesis. Such macromolecules may include polymers, polypeptides, polypeptoids, and nanoparticles. The macromolecules should be large enough to physically block access to the active site of the nucleotidyl transferase, not so large as to negatively alter the reaction kinetics. The macromolecules can be linked to nucleotide analogs using a variety of linking molecules, as described below.
In some embodiments, oligonucleotide synthesis may include introduction of a 3′ exonuclease to the one or more synthesized oligonucleotides after each nucleotide analog addition, but before cleaving the terminating group. The terminating group may block the 3′ exonuclease from acting on the oligonucleotides which have successfully added uncleaved nucleotide analogs, while oligonucleotides that have not successfully added the inhibitor will be removed by the 3′ exonuclease. In this manner, the invention allows for in-process quality control and may eliminate the need for post-synthesis purification.
Other aspects of the invention are apparent to the skilled artisan upon consideration of the following figures and detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a genus of deoxycytidine triphosphate (dCTP) analogs having a cleavable terminator linked at the N-4 position;
FIG. 1B shows cleavage of the cleavable terminator from a dCTP analog of FIG. 1A to achieve a “natural” dCTP and a cyclic leaving molecule;
FIG. 2A shows a genus of deoxyadenosine triphosphate (dATP) analogs having a cleavable terminator linked at the N-6 position;
FIG. 2B shows cleavage of the cleavable terminator from a dATP analog of FIG. 2A to achieve a “natural” dATP and a cyclic leaving molecule;
FIG. 3A shows a genus of deoxyguanosine triphosphate (dGTP) analogs having a cleavable terminator linked at the N-2 position;
FIG. 3B shows cleavage of the cleavable terminator from a dGTP analog of FIG. 3A to achieve a “natural” dGTP and a cyclic leaving molecule;
FIG. 4A shows a genus of deoxythymidine triphosphate (dTTP) analogs having a cleavable terminator linked at the N-3 position;
FIG. 4B shows cleavage of the cleavable terminator from a dTTP analog of FIG. 4A to achieve a “natural” dTTP and a cyclic leaving molecule;
FIG. 5A shows a genus of deoxyuridine triphosphate (dUTP) analogs having a cleavable terminator linked at the N-3 position;
FIG. 5B shows cleavage of the cleavable terminator from a dUTP analog of FIG. 5A to achieve a dUTP and a cyclic leaving molecule;
FIG. 6 shows an exemplary deoxycytidine triphosphate (dCTP) analog having a Staudinger linker connecting a blocking Asp-Asp molecule to the N-4 position of the deoxycytidine and subsequent cleavage of the Staudinger linker under aqueous conditions to achieve a dCTP and a leaving group;
FIG. 7A shows a genus of cytidine triphosphate (rCTP) analogs having a cleavable terminator linked at the N-4 position;
FIG. 7B shows cleavage of the cleavable terminator from a rCTP analog of FIG. 7A to achieve a “natural” rCTP and a cyclic leaving molecule;
FIG. 8A shows a genus of adenosine triphosphate (rATP) analogs having a cleavable terminator linked at the N-6 position;
FIG. 8B shows cleavage of the cleavable terminator from an rATP analog of FIG. 8A to achieve a “natural” rATP and a cyclic leaving molecule;
FIG. 9A shows n genus of guanosine triphosphate (rGTP) analogs having a cleavable terminator linked at the N-2 position;
FIG. 9B shows cleavage of the cleavable terminator from a rGTP analog of FIG. 9A to achieve a “natural” rGTP and a cyclic leaving molecule;
FIG. 10A shows a genus of thymidine triphosphate (rTTP) analogs having a cleavable terminator linked at the N-3 position;
FIG. 10B shows cleavage of the cleavable terminator from a rTTP analog of FIG. 10A to achieve a “natural” rTTP and a cyclic leaving molecule;
FIG. 11A shows a genus of uridine triphosphate (rUTP) analogs having a cleavable terminator linked at the N-3 position;
FIG. 11B shows cleavage of the cleavable terminator from a rUTP analog of FIG. 11A to achieve a rUTP and a cyclic leaving molecule;
FIG. 12 shows an exemplary cytidine triphosphate (rCTP) analog having a Staudinger linker connecting a blocking Asp-Asp molecule to the N-4 position of the cytidine and subsequent cleavage of the Staudinger linker under aqueous conditions to achieve a rCTP and a leaving group;
FIG. 13 shows an exemplary terminal deoxynucleotidyl transferase (TdT) mediated polynucleotide synthetic cycle, including: (a) incorporation of a nucleotide triphosphate analog comprising cleavable terminator, dN*TP—OH, and (b) removal of the terminating blocking group (indicated by *), thus enabling the next dN*TP—OH to be incorporated, wherein N=A, G, C, or T;
FIG. 14 shows an exemplary nucleotide analog with a cleavable linker comprising a variable number of methylene bridges;
FIG. 15 shows an exemplary nucleotide analog with a cleavable linker comprising a cysteine residue;
FIG. 16A shows an exemplary nucleotide analog with an anionic inhibitor comprising a single phosphate group;
FIG. 16B shows an exemplary nucleotide analog with an anionic inhibitor comprising two phosphate groups;
FIG. 16C shows an exemplary nucleotide analog with an anionic inhibitor comprising three phosphate groups;
FIG. 17 shows an exemplary microfluidic polynucleotide synthesis device;
FIG. 18 shows an exemplary polypeptoid inhibitor suitable for use in the invention;
FIG. 19 shows a flow-chart describing the use of a 3′ exonuclease to digest oligonucleotides that are not properly terminated between oligonucleotide synthesis cycles.



DETAILED DESCRIPTION
The invention provides improved methods for synthesizing polynucleotides, such as DNA and RNA, using enzymes and nucleic acid analogs. Using the disclosed methods, specific sequences of polynucleotides can be synthesized de novo, base by base, in an aqueous environment, without the use of a nucleic acid template. Additionally, because the nucleotide analogs have an unmodified 3′ hydroxyls, i.e., as found in “natural” deoxyribose and ribose molecules, the analogs result in “natural” nucleotides when an inhibitor, also referred to herein as a terminator or terminator group attached via a cleavable linker, is removed from the base. Other nucleotide analogs can also be used which, for example, include self-eliminating linkers, or nucleotides with modified phosphate groups. In most instances, the blocking group is designed to not leave behind substantial additional molecules, i.e., designed to leave behind “scarless” nucleotides that are recognized as “natural” nucleotides by the enzyme. Thus, at the conclusion of the synthesis, upon removal of the last blocking group, the synthesized polynucleotide is chemically and structurally equivalent to the naturally-occurring polynucleotide with the same sequence. The synthetic polynucleotide can, thus, be incorporated into living systems without concern that the synthesized polynucleotide will interfere with biochemical pathways or metabolism.
The process and analogs of the current invention can be used for the non-templated enzymatic synthesis of useful oligo- and oligodeoxynucleotides especially of long oligonucleotides (<5000 nt). Products can be single strand or partially double strand depending upon the initiator used. The synthesis of long oligonucleotides requires high efficiency incorporation and high efficiency of reversible terminator removal. The initiator bound to the solid support consists of a short, single strand DNA sequence that is either a short piece of the user defined sequence or a universal initiator from which the user defined single strand product is removed.
In one aspect, the disclosed methods employ commercially-available nucleotidyl transferase enzymes, such as terminal deoxynucleotidyl transferase (TdT), to synthesize polynucleotides from nucleotide analogs in a step-by-step fashion. The nucleotide analogs are of the form:

NTP-linker-inhibitor

wherein NTP is a nucleotide triphosphate (i.e., a dNTP or an rNTP), the linker is a cleavable linker between the pyridine or pyrimidine of the base, and the inhibitor is a group that prevents the enzyme from incorporating subsequent nucleotides. At each step, a new nucleotide analog is incorporated into the growing polynucleotide chain, whereupon the enzyme is blocked from adding an additional nucleotide by the inhibitor group. Once the enzyme has stopped, the excess nucleotide analogs can be removed from the growing chain, the inhibitor can be cleaved from the NTP, and new nucleotide analogs can be introduced in order to add the next nucleotide to the chain. By repeating the steps sequentially, it is possible to quickly construct nucleotide sequences of a desired length and sequence. Advantages of using nucleotidyl transferases for polynucleotide synthesis include: 1) 3′-extension activity using single strand (ss) initiating primers in a template-independent polymerization, 2) the ability to extend primers in a highly efficient manner resulting in the addition of thousands of nucleotides, and 3) the acceptance of a wide variety of modified and substituted NTPs as efficient substrates. In addition, the invention can make use of an initiator sequence that is a substrate for nucleotidyl transferase. The initiator is attached to a solid support and serves as a binding site for the enzyme. The initiator is preferably a universal initiator for the enzyme, such as a homopolymer sequence and is recyclable on the solid support, the formed oligonucleotide being cleavable from the initiator.

Methods of the invention are well-suited to a variety of applications that currently use synthetic nucleic acids, e.g., phosphoramidite-synthesized DNA oligos. For example, polynucleotides synthesized with the methods of the invention can be used as primers for nucleic acid amplification, hybridization probes for detection of specific markers, and for incorporation into plasmids for genetic engineering. However, because the disclosed methods produce longer synthetic strings of nucleotides, at a faster rate, and in an aqueous environment, the disclosed methods also lend themselves to high-throughput applications, such as screening for expression of genetic variation in cellular assays, as well as synthetic biology. Furthermore, the methods of the invention will provide the functionality needed for next-generation applications, such as using DNA as synthetic read/write memory, or creating macroscopic materials synthesized completely (or partially) from DNA.
The invention and systems described herein provide for synthesis of polynucleotides, including deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). While synthetic pathways for “natural” nucleotides, such as DNA and RNA, are described in the context of the common nucleic acid bases, e.g., adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), it is to be understood that the methods of the invention can be applied to so-called “non-natural” nucleotides, including nucleotides incorporating universal bases such as 3-nitropyrrole 2′-deoxynucleoside and 5-nitroindole 2′-deoxynucleoside, alpha phosphorothiolate, phosphorothioate nucleotide triphosphates, or purine or pyrimidine conjugates that have other desirable properties, such as fluorescence. Other examples of purine and pyrimidine bases include pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine. In some instances, it may be useful to produce nucleotide sequences having unreactive, but approximately equivalent bases, i.e., bases that do not react with other proteins, i.e., transcriptases, thus allowing the influence of sequence information to be decoupled from the structural effects of the bases.
Analogs
The invention provides nucleotide analogs having the formula NTP-linker-inhibitor for synthesis of polynucleotides in an aqueous environment. With respect to the analogs of the form NTP-linker-inhibitor, NTP can be any nucleotide triphosphate, such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate (TTP), uridine triphosphate (UTP), nucleotide triphosphates, deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP).
The linker can be any molecular moiety that links the inhibitor to the NTP and can be cleaved. For example, the linkers can be cleaved by adjusting the pH of the surrounding environment. The linkers may also be cleaved by an enzyme that is activated at a given temperature, but inactivated at another temperature. In some embodiments, the linkers include disulfide bonds.
Linkers may, for example, include photocleavable, nucleophilic, or electrophilic cleavage sites. Photocleavable linkers, wherein cleavage is activated by a particular wavelength of light, may include benzoin, nitroveratryl, phenacyl, pivaloyl, sisyl, 2-hydroxy-cinnamyl, coumarin-4-yl-methyl, or 2-nitrobenzyl based linkers.
Examples of nucleophilic cleavage sites include fluoride ion cleavable silicon-oxygen bonds or esters which may be cleaved in a basic solution. Electrophilically cleaved linkers may include acid induced cleavage sites which may comprise trityl, tert-butyloxycarbonyl groups, acetal groups, and p-alkoxybenzyl esters and amides. In certain aspects, a cleavable linker may include a cysteine residue as shown in FIG. 15.
The linker can be attached, for example, at the N4 of cytosine, the N3 or O4 of thymine, the N2 or N3 of guanine, and the N6 of adenine, or the N3 or O4 of uracil because attachment at a carbon results in the presence of a residual scar after removal of the polymerase-inhibiting group. The linker is typically on the order of at least about 10 Angstroms long, e.g., at least about 20 Angstroms long, e.g., at least about 25 Angstroms long, thus allowing the inhibitor to be far enough from the pyridine or pyrimidine to allow the enzyme to bind the NTP to the polynucleotide chain via the attached sugar backbone. In some embodiments, the cleavable linkers are self-cyclizing in that they form a ring molecule that is particularly non-reactive toward the growing nucleotide chain.
In certain aspects, a cleavable linker may include a variable number of methylene bridges on the NTP or the inhibitor side of a disulfide bond, including, for example, 1, 2, 3, or 4 methylene bridges as shown in FIGS. 14 and 16A-C. These methylene bridges may be used to increase the space between the NTP and the inhibitor. As noted above, the length of the cleavable linker may be selected in order to prevent the inhibitor from interfering with coupling of the NTP to the synthesized polynucleotide. In some embodiments of the invention, the distance of the charged group to the NTP plays an important role in the effectiveness of inhibiting a subsequent nucleotide incorporation.
For example, in some embodiments using a charged moiety as an inhibitor, the charged moiety may be from about 5 to about 60 bonds away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 40 bonds away from the NTP. In some other embodiments, the charged moiety of the inhibitor can be from about 10 to about 35 bonds away from the NTP. In some other embodiments, the charged moiety of the inhibitor may be from about 10 to about 30 bonds away from the NTP. In some other embodiments, the charged moiety of the inhibitor is from about 10 to about 20 bonds away from the NTP. The number of bonds between the charged moiety and the NTP may be increased by including additional methylene bridges.
The nucleotide analogs can include any moiety linked to the NTP that inhibits the coupling of subsequent nucleotides by the enzyme. The inhibitory group can be a charged group, such as a charged amino acid, or the inhibitory group can be a group that becomes charged depending upon the ambient conditions. In some embodiments, the inhibitor may include a moiety that is negatively charged or capable of becoming a negatively charged. For example, an inhibitor may include a chain of phosphate groups (e.g., 1, 2, or 3, phosphates) as shown in FIGS. 16A-C, wherein additional phosphates increase the overall anionic charge of the inhibitor. In other embodiments, the inhibitor group is positively charged or capable of becoming positively charged. In some other embodiments, the inhibitor is an amino acid or an amino acid analog. The inhibitor may be a peptide of 2 to 20 units of amino acids or analogs, a peptide of 2 to 10 units of amino acids or analogs, a peptide of 3 to 7 units of amino acids or analogs, a peptide of 3 to 5 units of amino acids or analogs. In some embodiments, the inhibitor includes a group selected from the group consisting of Glu, Asp, Arg, His, and Lys, and a combination thereof (e.g., Arg, Arg-Arg, Asp, Asp-Asp, Asp, Glu, Glu-Glu, Asp-Glu-Asp, Asp-Asp-Glu or AspAspAspAsp, etc.). Peptides or groups may be combinations of the same or different amino acids or analogs. In certain embodiments, a peptide inhibitor may be acetylated to discourage errant bonding of free amino groups. The inhibitory group may also include a group that reacts with residues in the active site of the enzyme thus interfering with the coupling of subsequent nucleotides by the enzyme. The inhibitor may have a charged group selected from the group consisting of —COO, —NO2, —PO4, —PO3, —SO2, or —NR3 where each R may be H or an alkyl group. In other embodiments, the inhibitor moiety does not comprise a —PO4 group.
In certain aspects, a terminator or inhibitor may include a steric inhibitor group. Such a steric inhibitor group may allow for the NTP-linker-inhibitor (i.e., nucleotide analog) to be incorporated onto the unblocked 3′ OH of an oligonucleotide, said incorporation being catalyzed by nucleotidyl transferase. The steric inhibitor group may physically block the incorporation of nucleotides or additional nucleotide analogs onto the unblocked 3′ OH of the incorporated nucleotide analog. Steric inhibitors may also block 3′ endonucleases from acting on a nucleotide analog and, accordingly, on oligonucleotides to which an un-cleaved nucleotide analog has been incorporated.
Steric inhibitors can include, for example, chemical polymers, nanoparticles, poly-N-substituted glycines (peptoids), or proteins. A steric inhibitor of the invention may be a variety of sizes including, e.g., greater than 20 Å, greater than 30 Å, greater than 40 Å, greater than 50 Å, greater than 60 Å, greater than 70 Å, greater than 80 Å, greater than 90 Å, greater than 100 Å, greater than 110 Å, greater than 120 Å, greater than 130 Å, greater than 140 Å, or greater than 150 Å. In preferred embodiments, a steric inhibitor may be monodisperse or substantially monodisperse. Steric inhibitors may be water soluble and conformationally constrained (i.e., of a rigid or semi-rigid form). In certain aspects, a steric inhibitor will physically block access to the active site of the relevant nucleotidyl transferase enzyme because of the size or the conformation of the inhibitor. In preferred embodiments, the steric inhibitor may comprise a non-natural bio-inspired polymer such as a polypeptoid or a non-natural polypeptide.
In certain aspects, a self-assembling polypeptoid sequence may be used as a steric inhibitor. Peptoid monomers are often based on an N-substituted glycine backbone. Because the backbone is devoid of hydrogen bond donors, polypeptoids are readily processed while still being able to form secondary structures such as helices. They also provide the beneficial properties of allowing polarities and side chains similar to peptides, while being generally chemically and thermally stable. Self-assembling polypeptoid steric inhibitors according to the invention may self-assemble single peptoid helices to form microspheres in the micrometer range of diameters including, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, or 3.5 μm, among others. In certain aspects, steric inhibitors may include peptoids with C-α-branched side chains, N-Aryl side chains, N-1-naphthylethyl side chains, or other formations capable of forming stable helical structures. An example of a peptoid steric inhibitor is shown in FIG. 18. FIG. 18 illustrates a branched poly-N-methoxyethyl glycine which may be used as a steric inhibitor according to the invention. In certain embodiments, a steric inhibitor may include a reactive group that is easily joined to a linker group, e.g., a cleavable linking group as described herein.
In other embodiments, a steric inhibitor may comprise a polymer, such as a biocompatible polymer. The polymer may comprise blocks of different polymers, such that the blocks form a desired macroscopic structure, e.g., a sphere when exposed to an aqueous environment. For example, the copolymer may comprise blocks of hydrophilic and hydrophobic blocks so that the polymer self-assembles into a micellar structure upon addition to water. In some embodiments, the hydrophobic blocks may be selected from polycaprolactones (PCL), polydimethylsiloxanes (PDMS), polymethylmethacrylate (PMMA), or polylactides (PLA). The hydrophilic blocks may include polyethylene glycol (PEG) or other polyalcohol.
In other embodiments, the inhibitor may comprise a nanoparticle of sufficient size to block the activity of a nucleotidyl transferase. Such nanoparticles may comprise, e.g., gold, silver, silicon, cerium oxide, iron oxide, titanium dioxide, silicon nitride, silicon boride, or silica, e.g., mesoporous silica. In other embodiments, the nanoparticles may comprise highly-ordered molecular structures, such as fullerenes, e.g., buckyballs and nanotubes, comprising carbon, or semiconductors.
Steric inhibitors may have no charge or may be positively or negatively charged to provide compatibility with the nucleotide to which it is linked and with the nucleotidyl transferase enzyme so that the inhibitor does not interfere with the incorporation reaction on the 5′ end of the NTP analog. Steric inhibitors may incorporate a variety of amino acid residues in order to provide a desired conformation, charge, or attachment site.
An example of a nucleotide analog of the type NTP-linker-inhibitor is shown in FIG. 1A. The analog in FIG. 1A includes an inhibitory (-Asp-Asp-) group linked to the N4 position of dCTP through a disulfide (—S—S—) bond while providing an unblocked, unmodified 3′-OH on the sugar ring. The linker is constructed such that all linker atoms (including the 2nd incorporation-inhibiting moiety) can be removed, thereby allowing the nascent DNA strand to revert to natural nucleotides. As shown in FIG. 1B, an aqueous reducing agent, such as tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), can be used to cleave the —S—S— bond, resulting in the loss of the inhibitor function (deblocking). As shown in FIG. 1B, a self-cyclizing linker can be incorporated, resulting in a cyclic oxidized tetrahydrothiophene leaving group that is easily removed from the reagent solution at the conclusion of nucleotide synthesis.
Exemplary schemes for synthesizing dCTP analogs of FIG. 1A are shown below in Schemes 1A and 1B:








In a fashion analogous to Schemes 1A and 1B, nucleotide analogs of the type NTP-linker-inhibitor can also be formed by attaching the linker-inhibitor moiety to the N6 of adenine (FIG. 2), the N2 of guanine (FIG. 3), the N3 of thymine (FIG. 4), or the N3 of uracil (FIG. 5), thereby providing analogs of the “naturally-occurring” dNTPs, as well as a deoxyuracil nucleotide (dUTP). While it is unlikely that there will be wide use of a dUTP, the synthesis is straightforward based upon the chemistry.
The invention is not limited to the linking chemistry of Schemes 1A and 1B, however, as carbamate, amide, or other self-eliminating linkages could also be employed. For example, nucleotides can also be prepared with Staudinger linkers, as shown in Scheme 2.




A deoxycytidine triphosphate (dCTP) analog created with a Staudinger linker (Scheme 2) to an Asp-Asp blocking group is shown in FIG. 6. As shown in FIG. 6, the Staudinger dCTP analog undergoes cleavage under aqueous conditions with the addition of azide and triphenylphosphine. The Staudinger analog shown in FIG. 6 is also suitable for nucleotide extension using nucleotidyl transferases, such as TdT, as described above and exemplified in FIGS. 1-5. While not shown explicitly in the FIGS., one of skill in the art can use Scheme 2 in conjunction with the suitable reactant to produce other nucleotide analogs having Staudinger linkers as needed for complete de novo nucleotide synthesis. In a fashion analogous to FIG. 6, nucleotide analogs of Scheme 2 can be formed by attaching the Staudinger moiety to the N6 of adenine, the N2 of guanine, the N3 of thymine, or the N3 of uracil, thereby providing analogs of the “naturally-occurring” dNTPs, as well as a deoxyuracil nucleotide (dUTP).
The methodologies of Scheme 1A can be used to produce corresponding ribonucleotide analogs, e.g., as shown in FIGS. 7-10, by starting with the appropriate ribonucleotide reactants. Ribonucleotide analogs comprising the Staudinger linker can also be created using Scheme 2 in order to form the needed ribonucleotide analogs, including, e.g., CTP analogs, as shown in FIG. 12. Furthermore, all of the ribonucleotide analogs, i.e., C, A, T, G, U, can be formed using a reaction similar to Scheme 2.
Enzymes
The methods of the invention employ nucleotidyl transferases to assemble the nucleotide analogs into polynucleotides. Nucleotidyl transferases include several families of related transferase and polymerase enzymes. Some nucleotidyl transferases polymerize deoxyribonucleotides more efficiently than ribonucleotides, some nucleotidyl transferases polymerize ribonucleotides more efficiently than deoxyribonucleotides, and some nucleotidyl transferases polymerize ribonucleotides and deoxyribonucleotides at approximately the same rate.
Of particular import to the invention, transferases having polymerase activity, such as terminal deoxynucleotidyl transferase (TdT), are capable of catalyzing the addition of deoxyribonucleotides to the 3′ end of a nucleotide chain, thereby increasing chain length in DNA nucleotides. TdT will only catalyze the addition of 1-2 ribonucleotides to the growing end of a DNA strand which could be useful in the construction of site specific DNA-RNA chimeric polynucleotides. In particular, calf thymus TdT, sourced from engineered E. coli, is suitable for use with the invention and available from commercial sources such as Thermo Scientific (Pittsburgh, Pa.). The amino acid sequence corresponding to calf TdT is listed in Table 1 as SEQ ID NO. 1.







TABLE 1 





Amino Acid Sequence of Bovine TdT















SEQ ID NO. 1: MAQQRQHQRL PMDPLCTASS GPRKKRPRQV


 


GASMASPPHD IKFQNLVLFI LEKKMGTTRR NFLMELARRK 


 


GFRVENELSD SVTHIVAENN SGSEVLEWLQ VQNIRASSQL 


 


ELLDVSWLIE SMGAGKPVEI TGKHQLVVRT DYSATPNPGF 


 


QKTPPLAVKK ISQYACQRKT TLNNYNHIFT DAFEILAENS


 


EFKENEVSYV TFMRAASVLK SLPFTIISMK DTEGIPCLGD 


 


KVKCIIEEII EDGESSEVKA VLNDERYQSF KLFTSVFGVG 


 


LKTSEKWFRM GFRSLSKIMS DKTLKFTKMQ KAGFLYYEDL 


 


VSCVTRAEAE AVGVLVKEAV WAFLPDAFVT MTGGFRRGKK


 


IGHDVDFLIT SPGSAEDEEQ LLPKVINLWE KKGLLLYYDL 


 


VESTFEKFKL PSRQVDTLDH FQKCFLILKL HHQRVDSSKS 


 


NQQEGKTWKA IRVDLVMCPY ENRAFALLGW TGSRQFERDI 


 


RRYATHERKM MLDNHALYDK TKRVFLKAES EEEIFAHLGL 


 


DYIEPWERNA









The nucleotide sequence corresponding to calf TdT is listed in Table 2 as SEQ ID NO. 2.








TABLE 2 





Nucleic Acid Sequence of Bovine TdT















SEQ ID NO. 2: ctcttctgga gataccactt gatggcacag


 


cagaggcagc atcagcgtct tcccatggat ccgctgtgca 


 


cagcctcctc aggccctcgg aagaagagac ccaggcaggt 


 


gggtgcctca atggcctccc ctcctcatga catcaagttt 


 


caaaatttgg tcctcttcat tttggagaag aaaatgggaa 


 


ccacccgcag aaacttcctc atggagctgg ctcgaaggaa


 


aggtttcagg gttgaaaatg agctcagtga ttctgtcacc 


 


cacattgtag cagaaaacaa ctctggttca gaggttctcg 


 


agtggcttca ggtacagaac ataagagcca gctcgcagct 


 


agaactcctt gatgtctcct ggctgatcga aagtatggga


 


gcaggaaaac cagtggagat tacaggaaaa caccagcttg 


 


ttgtgagaac agactattca gctaccccaa acccaggctt


 


ccagaagact ccaccacttg ctgtaaaaaa gatctcccag 


 


tacgcgtgtc aaagaaaaac cactttgaac aactataacc


 


acatattcac ggatgccttt gagatactgg ctgaaaattc 


 


tgagtttaaa gaaaatgaag tctcttatgt gacatttatg 


 


agagcagctt ctgtacttaa atctctgcca ttcacaatca 


 


tcagtatgaa ggatacagaa ggaattccct gcctggggga 


 


caaggtgaag tgtatcatag aggaaattat tgaagatgga 


 


gaaagttctg aagttaaagc tgtgttaaat gatgaacgat 


 


atcagtcctt caaactcttt acttctgttt ttggagtggg 


 


actgaagaca tctgagaaat ggttcaggat ggggttcaga 


 


tctctgagta aaataatgtc agacaaaacc ctgaaattca 


 


caaaaatgca gaaagcagga tttctctatt atgaagacct 


 


tgtcagctgc gtgaccaggg ccgaagcaga ggcggttggc 


 


gtgctggtta aagaggctgt gtgggcattt ctgccggatg 


 


cctttgtcac catgacagga ggattccgca ggggtaagaa 


 


gattgggcat gatgtagatt ttttaattac cagcccagga 


 


tcagcagagg atgaagagca acttttgcct aaagtgataa 


 


acttatggga aaaaaaggga ttacttttat attatgacct 


 


tgtggagtca acatttgaaa agttcaagtt gccaagcagg 


 


caggtggata ctttagatca ttttcaaaaa tgctttctga 


 


ttttaaaatt gcaccatcag agagtagaca gtagcaagtc


 


caaccagcag gaaggaaaga cctggaaggc catccgtgtg 


 


gacctggtta tgtgccccta cgagaaccgt gcctttgccc 


 


tgctaggctg gactggctcc cggcagtttg agagagacat 


 


ccggcgctat gccacacacg agcggaagat gatgctggat 


 


aaccacgctt tatatgacaa gaccaagagg gtatttctca 


 


aagcggaaag tgaagaagaa atctttgcac atctgggatt 


 


ggactacatt gaaccatggg aaagaaatgc ttaggagaaa 


 


gctgtcaact tttttctttt ctgttctttt tttcaggtta 


 


gacaaattat gcttcatatt ataatgaaag atgccttagt 


 


caagtttggg attctttaca ttttaccaag atgtagattg 


 


cttctagaaa taagtagttt tggaaacgtg atcaggcacc 


 


ccctgggtta tgctctggca agccatttgc aggactgatg 


 


tgtagaactc gcaatgcatt ttccatagaa acagtgttgg 


 


aattggtggc tcatttccag ggaagttcat caaagcccac 


 


tttgcccaca gtgtagctga aatactgtat acttgccaat 


 


aaaaatagga aac









While commercially-available TdT is suitable for use with the methods of the invention, modified TdT, e.g., having an amino acid sequence at least 95% in common with SEQ ID NO. 1, e.g., having an amino acid sequence at least 98% in common with SEQ ID NO. 1, e.g., having an amino acid sequence at least 99% in common with SEQ ID NO. 1, may be used with the methods of the invention. An organism that expresses a suitable nucleotidyl transferase may comprise a nucleic acid sequence at least 95% in common with SEQ ID NO. 2, e.g., at least 98% in common with SEQ ID NO. 2, e.g., at least 99% in common with SEQ ID NO. 2. In some instances, a modified TdT will result in more efficient generation of polynucleotides, or allow better control of chain length. Other modifications to the TdT may change the release characteristics of the enzyme, thereby reducing the need for aqueous reducing agents such as TCEP or DTT.
For the synthesis of RNA polynucleotides, a nucleotidyl transferase like E. coli poly(A) polymerase can be used to catalyze the addition of ribonucleotides to the 3′ end of a ribonucleotide initiator. In other embodiments, E. coli poly(U) polymerase may be more suitable for use with the methods of the invention. Both E. coli poly(A) polymerase and E. coli poly(U) polymerase are available from New England Biolabs (Ipswich, Mass.). These enzymes may be used with 3′unblocked reversible terminator ribonucleotide triphosphates (rNTPs) to synthesize RNA. In certain embodiments, RNA may be synthesized using 3′blocked, 2′blocked, or 2′-3′blocked rNTPs and poly(U) polymerase or poly(A) polymerase. The amino acid and nucleotide sequences for E. coli Poly(A) polymerase and E. coli Poly(U) polymerase are reproduced below. Modified E. coli Poly(A) polymerase or E. coli Poly(U) polymerase may be suitable for use with the methods of the invention. For example, an enzyme, having an amino acid sequence at least 95% in common with SEQ ID NO. 3, e.g., having an amino acid sequence at least 98% in common with SEQ ID NO. 3, e.g., having an amino acid sequence at least 99% in common with SEQ ID NO. 3, may be used with the methods of the invention. An organism that expresses a suitable enzyme may comprise a nucleic acid sequence at least 95% in common with SEQ ID NO. 4, e.g., at least 98% in common with SEQ ID NO. 4, e.g., at least 99% in common with SEQ ID NO. 4. Alternatively, an enzyme having an amino acid sequence at least 95% in common with SEQ ID NO. 5, e.g., having an amino acid sequence at least 98% in common with SEQ ID NO. 5, e.g., having an amino acid sequence at least 99% in common with SEQ ID NO. 5, may be used with the methods of the invention. An organism that expresses a suitable enzyme may comprise a nucleic acid sequence at least 95% in common with SEQ ID NO. 6, e.g., at least 98% in common with SEQ ID NO. 6, e.g., at least 99% in common with SEQ ID NO. 6.







TABLE 3 





Amino Acid Sequence of E. coli Poly(A) polymerase















SEQ ID NO. 3: MFTRVANFCR KVLSREESEA EQAVARPQVT


 


VIPREQHAIS RKDISENALK VMYRLNKAGY EAWLVGGGVR 


 


DLLLGKKPKD FDVTTNATPE QVRKLFRNCR LVGRRFRLAH 


 


VMFGPEIIEV ATFRGHHEGN VSDRTTSQRG QNGMLLRDNI 


 


FGSIEEDAQR RDFTINSLYY SVADFTVRDY VGGMKDLKDG 


 


VIRLIGNPET RYREDPVRML RAVRFAAKLG MRISPETAEP 


 


IPRLATLLND IPPARLFEES LKLLQAGYGY ETYKLLCEYH 


 


LFQPLFPTIT RYFTENGDSP MERIIEQVLK NTDTRIHNDM 


 


RVNPAFLFAA MFWYPLLETA QKIAQESGLT YHDAFALAMN 


 


DVLDEACRSL AIPKRLTTLT RDIWQLQLRM SRRQGKRAWK 


 


LLEHPKFRAA YDLLALRAEV ERNAELQRLV KWWGEFQVSA 


 


PPDQKGMLNE LDEEPSPRRR TRRPRKRAPR REGTA









The nucleotide sequence corresponding to E. coli poly(A) polymerase is listed in Table 4 as SEQ ID NO. 4.








TABLE 4 





Nucleotide Sequence of E. coli Poly(A) polymerase















SEQ ID NO. 4: atttttaccc gagtcgctaa tttttgccgc


 


aaggtgctaa gccgcgagga aagcgaggct gaacaggcag 


 


tcgcccgtcc acaggtgacg gtgatcccgc gtgagcagca 


 


tgctatttcc cgcaaagata tcagtgaaaa tgccctgaag 


 


gtaatgtaca ggctcaataa agcgggatac gaagcctggc 


 


tggttggcgg cggcgtgcgc gacctgttac ttggcaaaaa


 


gccgaaagat tttgacgtaa ccactaacgc cacgcctgag 


 


caggtgcgca aactgttccg taactgccgc ctggtgggtc 


 


gccgtttccg tctggctcat gtaatgtttg gcccggagat 


 


tatcgaagtt gcgaccttcc gtggacacca cgaaggtaac 


 


gtcagcgacc gcacgacctc ccaacgcggg caaaacggca 


 


tgttgctgcg cgacaacatt ttcggctcca tcgaagaaga 


 


cgcccagcgc cgcgatttca ctatcaacag cctgtattac 


 


agcgtagcgg attttaccgt ccgtgattac gttggcggca


 


tgaaggatct gaaggacggc gttatccgtc tgattggtaa 


 


cccggaaacg cgctaccgtg aagatccggt acgtatgctg 


 


cgcgcggtac gttttgccgc caaattgggt atgcgcatca 


 


gcccggaaac cgcagaaccg atccctcgcc tcgctaccct 


 


gctgaacgat atcccaccgg cacgcctgtt tgaagaatcg 


 


cttaaactgc tacaagcggg ctacggttac gaaacctata 


 


agctgttgtg tgaatatcat ctgttccagc cgctgttccc 


 


gaccattacc cgctacttca cggaaaatgg cgacagcccg 


 


atggagcgga tcattgaaca ggtgctgaag aataccgata 


 


cgcgtatcca taacgatatg cgcgtgaacc cggcgttcct 


 


gtttgccgcc atgttctggt acccactgct ggagacggca 


 


cagaagatcg cccaggaaag cggcctgacc tatcacgacg 


 


ctttcgcgct ggcgatgaac gacgtgctgg acgaagcctg 


 


ccgttcactg gcaatcccga aacgtctgac gacattaacc 


 


cgcgatatct ggcagttgca gttgcgtatg tcccgtcgtc 


 


agggtaaacg cgcatggaaa ctgctggagc atcctaagtt 


 


ccgtgcggct tatgacctgt tggccttgcg agctgaagtt 


 


gagcgtaacg ctgaactgca gcgtctggtg aaatggtggg 


 


gtgagttcca ggtttccgcg ccaccagacc aaaaagggat 


 


gctcaacgag ctggatgaag aaccgtcacc gcgtcgtcgt 


 


actcgtcgtc cacgcaaacg cgcaccacgt cgtgagggta 


 


ccgcatga
















TABLE 5 





Amino Acid Sequence of E. coli Poly(U) polymerase















SEQ ID NO. 5: GSHMSYQKVP NSHKEFTKFC YEVYNEIKIS


 


DKEFKEKRAA LDTLRLCLKR ISPDAELVAF GSLESGLALK 


 


NSDMDLCVLM DSRVQSDTIA LQFYEELIAE GFEGKFLQRA 


 


RIPIIKLTSD TKNGFGASFQ CDIGFNNRLA IHNTLLLSSY 


 


TKLDARLKPM VLLVKHWAKR KQINSPYFGT LSSYGYVLMV 


 


LYYLIHVIKP PVFPNLLLSP LKQEKIVDGF DVGFDDKLED 


 


IPPSQNYSSL GSLLHGFFRF YAYKFEPREK VVTFRRPDGY 


 


LTKQEKGWTS ATEHTGSADQ IIKDRYILAI EDPFEISHNV 


 


GRTVSSSGLY RIRGEFMAAS RLLNSRSYPI PYDSLFEEA









The nucleotide sequence corresponding to E. coli poly(U) polymerase is listed in Table 6 as SEQ ID NO. 6.








TABLE 6 





Nucleotide Sequence of E. coli Poly(A) polymerase















SEQ ID NO. 6: ggcagccata tgagctatca gaaagtgccg


 


aacagccata aagaatttac caaattttgc tatgaagtgt 


 


ataacgaaat taaaattagc gataaagaat ttaaagaaaa 


 


acgcgcggcg ctggataccc tgcgcctgtg cctgaaacgc 


 


attagcccgg atgcggaact ggtggcgttt ggcagcctgg 


 


aaagcggcct ggcgctgaaa aacagcgata tggatctgtg 


 


cgtgctgatg gatagccgcg tgcagagcga taccattgcg 


 


ctgcagtttt atgaagaact gattgcggaa ggctttgaag 


 


gcaaatttct gcagcgcgcg cgcattccga ttattaaact 


 


gaccagcgat accaaaaacg gctttggcgc gagctttcag 


 


tgcgatattg gctttaacaa ccgcctggcg attcataaca 


 


ccctgctgct gagcagctat accaaactgg atgcgcgcct 


 


gaaaccgatg gtgctgctgg tgaaacattg ggcgaaacgc 


 


aaacagatta acagcccgta ttttggcacc ctgagcagct 


 


atggctatgt gctgatggtg ctgtattatc tgattcatgt 


 


gattaaaccg ccggtgtttc cgaacctgct gctgagcccg 


 


ctgaaacagg aaaaaattgt ggatggcttt gatgtgggct 


 


ttgatgataa actggaagat attccgccga gccagaacta 


 


tagcagcctg ggcagcctgc tgcatggctt ttttcgcttt 


 


tatgcgtata aatttgaacc gcgcgaaaaa gtggtgacct 


 


ttcgccgccc ggatggctat ctgaccaaac aggaaaaagg 


 


ctggaccagc gcgaccgaac ataccggcag cgcggatcag 


 


attattaaag atcgctatat tctggcgatt gaagatccgt 


 


ttgaaattag ccataacgtg ggccgcaccg tgagcagcag 


 


cggcctgtat cgcattcgcg gcgaatttat ggcggcgagc 


 


cgcctgctga acagccgcag ctatccgatt ccgtatgata 


 


gcctgtttga agaagcg









As discussed above, the inhibitor coupled to the nucleotide analog will cause the transferase, e.g., TdT, to not release from the polynucleotide or prevent other analogs from being incorporated into the growing chain. A charged moiety results in better inhibition, however, research suggests that the specific chemical nature of the inhibitor is not particularly important. For example, both phosphates and acidic peptides can be used to inhibit enzymatic activity. See, e.g., Bowers et al., Nature Methods, vol. 6, (2009) p. 593-95, and U.S. Pat. No. 8,071,755, both of which are incorporated herein by reference in their entireties. In some embodiments, the inhibitor will include single amino acids or dipeptides, like -(Asp)2, however the size and charge on the moiety can be adjusted, as needed, based upon experimentally determined rates of first nucleotide incorporation and second nucleotide incorporation. That is, other embodiments may use more or different charged amino acids or other biocompatible charged molecule.
Other methods of nucleotide synthesis may be used to build de novo oligonucleotides in a template independent fashion using nucleotidyl transferases or modified nucleotidyl transferases. In one embodiment, the polymerase/transferase enzymes can be modified so that they cease nucleotide addition when they encounter a modification to the phosphate of a 3′-unmodified dNTP analog. This scheme would require a deblocking reagent/reaction that modifies the phosphate end of the nucleotide analog, which frees up the nascent strand for subsequent nucleotide incorporation. Preferred embodiments of this approach would use nucleotide analogs modified only at the phosphates (alpha, beta or gamma) although modifications of the purine/pyrimidine base of the nucleotide are allowed.
In some embodiments, it may be advantageous to use a 3′ exonuclease to remove oligonucleotides that have not been properly terminated with an inhibitor prior to subsequent nucleotide analog addition. In particular, the inhibitor of the nucleotide analog can be chosen to inhibit the activity of nucleotidyl transferase and 3′ exonucleases, such that only properly terminated oligonucleotides would be built up. Using this quality control technique, the purity of the resulting oligonucleotide sequences would be improved. In some embodiments, use of such quality control measures can negate the need for post-synthesis purification. This technique is represented schematically in FIG. 19, where a 3′ exonuclease is introduced after a wash step to remove excess nucleotide analogs and prior to linker cleavage. Such a cleaning step, as shown in FIG. 19, will reduce the number of oligonucleotides that are of an undesired length and/or sequence.
Another embodiment for using non-template dependent polymerase/transferase enzymes would be to using protein engineering or protein evolution to modify the enzyme to remain tightly bound and inactive to the nascent strand after each single nucleotide incorporation, thus preventing any subsequent incorporation until such time as the polymerase/transferase is released from the strand by use of a releasing reagent/condition. Such modifications would be selected to allow the use of natural unmodified dNTPs instead of reversible terminator dNTPs. Releasing reagents could be high salt buffers, denaturants, etc. Releasing conditions could be high temperature, agitation, etc. For instance, mutations to the Loop1 and SD1 regions of TdT have been shown to dramatically alter the activity from a template-independent activity to more of a template dependent activity. Specific mutations of interest include but are not limited to Δ3384/391/392, del loop1 (386→398), L398A, D339A, F401A, and Q402K403C404→E402R403S404. Other means of accomplishing the goal of a post-incorporation tight binding (i.e., single turnover) TdT enzyme could include mutations to the residues responsible for binding the three phosphates of the initiator strand including but not limited to K261, R432, and R454.
Another embodiment for using non-template dependent polymerase/transferase enzymes would be to use protein engineering or protein evolution to modify the enzyme to accept 3-blocked reversible terminators with high efficiency. Most naturally occurring polymerase/transferase enzymes will not incorporate 3′-blocked reversible terminators due to steric constraints in the active site of the enzyme. Modifying either single or several aa residues in the active site of the enzyme can allow the highly efficient incorporation of 3′-blocked reversible terminators into a support bound initiator in a process completely analogous to that described above. After incorporation, the 3′-reversible terminator is removed with a deblocking reagent/condition thus generating a completely natural (scarless) single strand molecule ready for subsequent controlled extension reactions. There are residues close to the 3′-OH of the incoming dNTP which explains the propensity of TdT for incorporating ribonucleotide triphosphates as readily as deoxyribonucleotide triphosphates; residues including but not limited to those between β1 and β2 especially R334, Loop1, and those between α13 and α14, especially R454, are likely targets for mutagenesis to accommodate the bulk of 3′-reversible terminator groups and allow their efficient incorporation. In certain embodiments additional amino acid changes may be required to compensate for these residue alterations made to accommodate a 3′-reversible terminator. Another embodiment for using template-dependent polymerases would be to use the either 3′blocked or 3′unblocked dNTP analogs with a plurality of primer-template pairs attached to a solid support where the template is a nucleic acid analog that supports polymerase mediated primer extension of any of the four bases as specified by the user.
Another embodiment for using non-template dependent polymerase/transferase enzymes can use protein engineering or protein evolution to modify the enzyme to optimize the use of each of the four different nucleotides or even different modified nucleotide analogs in an analog specific manner. Nucleotide specific or nucleotide analog specific enzyme variants could be engineered to possess desirable biochemical attributes like reduced Km or enhanced addition rate which would further reduce the cost of the synthesis of desired polynucleotides.
Solid State Synthesis
The methods of the invention can be practiced under a variety of reaction conditions, however the orderly construction and recovery of desired polynucleotides will, in most cases, require a solid support to which the polynucleotides can be grown. In some embodiments, the methods include the enzymatically-mediated synthesis of polynucleotides on a solid support. When used in conjunction with the NTP, linker, and inhibitor analogs discussed above, it is possible to construct specific polynucleotide sequences of DNA as well as RNA by using, for example, TdT or poly(A) polymerase in an aqueous environment. As shown in FIG. 13, the TdT can be used to effect the stepwise construction of custom polynucleotides by extending the polynucleotide sequence a stepwise fashion. As discussed previously, the inhibitor group of each NTP analog causes the enzyme to stop with the addition of a nucleotide. After each nucleotide extension step, the reactants are washed away from the solid support prior to the removal of the inhibitor by cleaving the linker, and then new reactants are added, allowing the cycle to start anew.
In certain embodiments, an additional quality control step may be incorporated in which the oligonucleotide or polynucleotide sequence is exposed to 3′ exonuclease after the nucleotidyl transferase mediated nucleotide analog extension step and before inhibitor cleavage. 3′ exonuclease may degrade oligonucleotide or polynucleotide strands with an unblocked 3′ OH. In certain aspects, an uncleaved inhibitor (e.g., a steric inhibitor) may physically block the 3′ exonuclease from degrading a strand to which an uncleaved nucleotide analog has been successfully incorporated. Such a quality control step may degrade only the oligonucleotides or polynucleotides which have unsuccessfully incorporated the desired nucleotide analog, thereby eliminating any errors in the finished synthesized sequence potentially removing the need for post-synthesis sequence verification. After 3′ exonuclease exposure, the enzyme may be washed away before carrying on with the inhibitor cleavage step.
The 3′ exonuclease may act by shortening or completely degrading strands that have not successfully added the desired nucleotide analog. Strands that fail to be enzymatically extended at a given cycle will not have a terminal macromolecule-dNMP conjugate prior to the linker cleavage step. If a 3′-exonuclease is introduced at this stage, the full length strand could be protected from degradation while “failure” strands will be shorted in length or potentially degraded completely to mononucleotide phosphates. The yield of long (>500 bases) synthetic DNA is dependent on highly efficient reactions occurring at each and every cycle; both the enzymatic extension and the deblocking/self-elimination steps must occur at near quantitative yields. The introduction of a 3′-exonuclease after the enzymatic extension step but before the macromolecule terminator cleavage step would have a positive impact on the nascent strand purity if the extension efficiency is less than quantitative (i.e., there are strands that are not extended and therefore possess a natural unmodified terminal nucleotide).
*Conversely, a 3′-exonuclease step would have no impact on the quality of the synthesis if the deblocking/elimination step is less than quantitative because those strands would still be protected by the macromolecule terminator and fail to extend during the next extension step. Thus the actual improvement of the quality of the synthesis with the addition of a 3′-exonuclease step can only be experimentally determined and then an assessment made if it is worth the additional cost and cycle time. Since much of the cost of long ODNs made by the existing phosphoramidite method is related to post-synthesis
At the conclusion of n cycles of extension-remove-deblocking-wash, the finished full-length, single-strand polynucleotide is complete and can be cleaved from the solid support and recovered for subsequent use in applications such as DNA sequencing or PCR. Alternatively, the finished, full-length, single-strand polynucleotide can remain attached to the solid support for subsequent use in applications such as hybridization analysis, protein or DNA affinity capture. In other embodiments, partially double-stranded DNA can be used as an initiator, resulting in the synthesis of double-stranded polynucleotides.
Solid supports suitable for use with the methods of the invention may include glass and silica supports, including beads, slides, pegs, or wells. In some embodiments, the support may be tethered to another structure, such as a polymer well plate or pipette tip. In some embodiments, the solid support may have additional magnetic properties, thus allowing the support to be manipulated or removed from a location using magnets. In other embodiments, the solid support may be a silica coated polymer, thereby allowing the formation of a variety of structural shapes that lend themselves to automated processing.
Synthesizers
To capitalize on the efficiency of the disclosed methods, an aqueous phase DNA synthesizer can be constructed to produce desired polynucleotides in substantial quantities. In one embodiment, a synthesizer will include four wells of the described NTP analog reagents, i.e., dCTP, dATP, dGTP, and dTTP, as well as TdT at concentrations sufficient to effect polynucleotide growth. A plurality of initiating sequences can be attached to a solid support that is designed to be repeatedly dipped into each of the four wells, e.g., using a laboratory robot. The robot could be additionally programmed to rinse the solid support in wash buffer between nucleotide additions, cleave the linking group by exposing the support to a deblocking agent, and wash the solid support a second time prior to moving the solid support to the well of the next desired nucleotide. With simple programming, it is possible to create useful amounts of desired nucleotide sequences in a matter of hours, and with substantial reductions hazardous waste. Ongoing synthesis under carefully controlled conditions will allow the synthesis of polynucleotides with lengths in the thousands of base pairs. Upon completion, the extension products are released from the solid support, whereupon they can be used as finished nucleotide sequences.
A highly parallel embodiment could consist of a series of initiator-solid supports on pegs in either 96 or 384 well formats that could be individually retracted or lowered so that the pegs can be indexed to contact the liquids in the wells in a controlled fashion. The synthesizer could thus consist of the randomly addressable peg device, four enzyme-dNTP analog reservoirs in the same format as the peg device (96 or 384 spacing), additional reagent reservoirs (washing, deblocking, etc.) in the same format as the peg device (96 or 384 spacing), and a transport mechanism (e.g., a laboratory robot) for moving the peg device from one reservoir to another in a user programmable controlled but random access fashion. Care must be taken to avoid contaminating each of the four enzyme-dNTP reservoirs since the contents are reused throughout the entire synthesis process to reduce the cost of each polynucleotide synthesis.
In alternative embodiments, the reagents (e.g., nucleotide analogs, enzymes, buffers) will be moved between solid supports, allowing the reagents to be recycled. For example a system of reservoirs and pumps can move four different nucleotide analog solutions, wash buffers, and/or reducing agent solutions between one or more reactors in which the oligonucleotides will be formed. The reactors and pumps can be conventional, or the devices may be constructed using microfluidics. Because of the non-anhydrous (aqueous) nature of the process, no special care needs to be taken in the design of the hardware used to eliminate exposure to water. The synthesis process can take place with only precautions to control evaporative loss. A highly parallel embodiment could consist of a monolithic series of initiator-solid supports on pegs in either 96 or 384 well format that can be interfaced to a series of wells in the same matching format. Each well would actually be a reaction chamber that is fed by four enzyme-dNTP analog reservoirs and additional reagent reservoirs (washing, deblocking, etc.) with appropriate valves. Provisions would be made in the fluidics logic to recover the enzyme-dNTP reactants in a pristine fashion after each extension reaction since they are reused throughout the entire synthesis process to reduce the cost of each polynucleotide synthesis. In other embodiments, a system of pipetting tips could be used to add and remove reagents.
In certain aspects, polynucleotides may be synthesized using microfluidic devices and/or inkjet printing technology. An exemplary microfluidic polynucleotide synthesis device is shown in FIG. 17 for illustrative purposes and not to scale. Microfluidic channels 255, including regulators 257, couple reservoirs 253 to a reaction chamber 251 and an outlet channel 259, including a regulator 257 can evacuate waste from the reaction chamber 251. Microfluidic devices for polynucleotide synthesis may include, for example, channels 255, reservoirs 253, and/or regulators 257. Polynucleotide synthesis may occur in a microfluidic reaction chamber 251 which may include a number of anchored synthesized nucleotide initiators which may include beads or other substrates anchored or bound to an interior surface of the reaction chamber and capable of releasably bonding a NTP analog or polynucleotide initiator. The reaction chamber 251 may include at least one intake and one outlet channel 259 so that reagents may be added and removed to the reaction chamber 251. The microfluidic device may include a reservoir 253 for each respective NTP analog. Each of these NTP analog reservoirs 253 may also include an appropriate amount of TdT or any other enzyme which elongates DNA or RNA strands without template direction. Additional reservoirs 253 may contain reagents for linker/inhibitor cleavage and washing. These reservoirs 253 can be coupled to the reaction chamber 251 via separate channels 255 and reagent flow through each channel 255 into the reaction chamber 251 may be individually regulated through the use of gates, valves, pressure regulators, or other means. Flow out of the reaction chamber 251, through the outlet channel 259, may be similarly regulated.
In certain instances, reagents may be recycled, particularly the NTP analog-enzyme reagents. Reagents may be drawn back into their respective reservoirs 253 from the reaction chamber 251 via the same channels 255 through which they entered by inducing reverse flow using gates, valves, pressure regulators or other means. Alternatively, reagents may be returned from the reaction chamber 251 to their respective reservoirs 253 via independent return channels. The microfluidic device may include a controller capable of operating the gates, valves, pressure, or other regulators 257 described above.
An exemplary microfluidic polynucleotide synthesis reaction may include flowing a desired enzyme-NTP analog reagent into the reaction chamber 251; after a set amount of time, removing the enzyme-NTP analog reagent from the reaction chamber 251 via an outlet channel 259 or a return channel; flowing a wash reagent into the reaction chamber 251; removing the wash reagent from the reaction chamber 251 through an outlet channel 259; flowing a de-blocking or cleavage reagent into the reaction chamber 251; removing the de-blocking or cleavage reagent from the reaction chamber 251 via an outlet channel 259 or a return channel; flowing a wash reagent into the reaction chamber 251; removing the wash reagent from the reaction chamber 251 through an outlet channel 259; flowing the enzyme-NTP analog reagent including the next NTP in the desired sequence to be synthesized into the reaction chamber 251; and repeating until the desired polynucleotide has been synthesized. After the desired polynucleotide has been synthesized, it may be released from the reaction chamber anchor or substrate and collected via an outlet channel 259 or other means.
In certain aspects, reagents and compounds, including NTP analogs, TdT and/or other enzymes, and reagents for linker/inhibitor cleavage and/or washing may be deposited into a reaction chamber using inkjet printing technology or piezoelectric drop-on-demand (DOD) inkjet printing technology. Inkjet printing technology can be used to form droplets, which can be deposited, through the air, into a reaction chamber. Reagent droplets may have volumes in the picoliter to nanoliter scale. Droplets may be introduced using inkjet printing technology at a variety of frequencies including 1 Hz, 10, Hz, 100 Hz, 1 kHz, 2 kHz, and 2.5 kHz. Various reagents may be stored in separate reservoirs within the inkjet printing device and the inkjet printing device may deliver droplets of various reagents to various discrete locations including, for example, different reaction chambers or wells within a chip. In certain embodiments, inkjet and microfluidic technologies may be combined wherein certain reagents and compounds are delivered to the reaction chamber via inkjet printing technology while others are delivered via microfluidic channels or tubes. An inkjet printing device may be controlled by a computing device comprising at least a non-transitory, tangible memory coupled to a processor. The computing device may be operable to receive input from an input device including, for example, a touch screen, mouse, or keyboard and to control when and where the inkjet printing device deposits a droplet of reagent, the reagent it deposits, and/or the amount of reagent deposited.
In certain instances, a desired polynucleotide sequence may be entered into the computing device through an input device wherein the computing device is operable to perform the necessary reactions to produce the desired polynucleotide sequence by sequentially depositing the appropriate NTP analog, enzyme, cleavage reagent, and washing reagent, in the appropriate order as described above.
After synthesis, the released extension products can to be analyzed by high resolution PAGE to determine if the initiators have been extended by the anticipated number of bases compared to controls. A portion of the recovered synthetic DNA may also be sequenced to determine if the synthesized polynucleotides are of the anticipated sequence.
Because the synthesizers are relatively simple and do not require the toxic components needed for phosphoramidite synthesis, synthesizers of the invention will be widely accessible for research institutions, biotechs, and hospitals. Additionally, the ability to reuse/recycle reagents will reduce the waste produced and help reduce the costs of consumables. The inventors anticipate that the methods and systems will be useful in a number of applications, such as DNA sequencing, PCR, and synthetic biology.
INCORPORATION BY REFERENCE
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
EQUIVALENTS
Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

## Claims

1. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of either a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor is a macromolecule greater than 50 Å in diameter.

2. The method of claim 1, wherein the nucleotide analog comprises the following structure:

3. The method of claim 1, wherein the nucleotide analog comprises the following structure:

4. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor comprises the following structure:

5. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor comprises a polypeptoid.

6. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor comprises a polymer.

7. The method of claim 6, wherein the polymer comprises polyethylene glycol.

8. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor is a protein.

9. A method for synthesizing an oligonucleotide, comprising:
exposing an oligonucleotide attached to a solid support to a nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template such that the nucleotide analog is incorporated into the oligonucleotide,
wherein the nucleotide analog comprises a nucleotide coupled, by a cleavable linker, to an inhibitor that sterically prevents the nucleotidyl transferase from catalyzing incorporation of a natural nucleotide or a nucleotide analog into said oligonucleotide until said inhibitor is removed by cleavage of said cleavable linker, and
wherein the inhibitor is a nanoparticle.

10. The method of claim 1, wherein the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution having a pH between about 6.5 and 8.5.

11. The method of claim 1, the nucleic acid attached to the solid support is exposed to the nucleotide analog in the presence of an aqueous solution at a temperature between about 35 and 39° C.

12. The method of claim 1, wherein the solid support is a bead, a well, or a peg.

13. The method of claim 1, wherein the nucleic acid is single stranded.

14. The method of claim 1, wherein the cleavable linker comprises a moiety that forms a cyclic by-product when cleaved from the nucleotide analog.

15. The method of claim 1, further comprising:
cleaving the cleavable linker in order to produce a native nucleotide; and
exposing the native nucleotide to a second nucleotide analog in the presence of a nucleotidyl transferase enzyme and in the absence of a nucleic acid template.

16. The method of claim 1, further comprising exposing the oligonucleotide to an exonuclease enzyme.

17. The method of claim 1, further comprising providing an aqueous solution comprising the nucleotide analog and the nucleotidyl transferase enzyme.

18. The method of claim 1, wherein the inhibitor comprises a charged moiety.

19. The method of claim 1, wherein the nucleotide analog comprises a ribose sugar or a deoxyribose sugar.

20. The method of claim 1, wherein the nucleotide substrate comprises a base selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil.

21. The method of claim 1, wherein the nucleotidyl transferase comprises a protein sequence that is at least about 90% identical to SEQ ID NO. 1, SEQ ID NO. 3, or SEQ ID NO. 5.

22. The method of claim 1, wherein the nucleotidyl transferase originates from an organism having a nucleotide sequence that is at least about 90% identical to SEQ ID NO. 2, SEQ ID NO. 4, or SEQ ID NO. 6.

23. The method of claim 1, wherein the nucleotide analog is delivered in a droplet.