# Patent

## Title
Functional ligands to target molecules

## Abstract

The present invention relates functional ligands to target molecules, particularly to functional nucleic acids and modifications thereof, and to methods for simultaneously generating, for example, numerous different functional biomolecules, particularly to methods for generating numerous different functional nucleic acids against multiple target molecules simultaneously. The present invention further relates to functional ligands which bind with affinity to target molecules. The present invention further relates to methods for generating, for example, functional biomolecules, particularly to functional nucleic acids, that bind with functional activity to another biomolecule, such as a receptor molecule. More than one or multiple targets as used herein may generally include different types of targets, and/or may also include a multitude of a singular type of targets at different conditions, such as, for example, temperature, pH, chemical environment, and/or any other appropriate conditions.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. utility patent application Ser. No. 13/493,996, filed Jun. 11, 2012, entitled “FUNCTIONAL LIGANDS TO TARGET MOLECULES”, which is still pending, which is a non-provisional of U.S. provisional patent application Ser. No. 61/495,976, entitled “FUNCTIONAL LIGANDS TO TARGET MOLECULES”, filed Jun. 11, 2011, and is a continuation-in-part of U.S. utility patent application Ser. No. 12/683,429, filed Jan. 7, 2010, entitled “METHODS FOR SIMULTANEOUS GENERATION OF FUNCTIONAL LIGANDS”, which issued as U.S. Pat. No. 8,314,052 on Nov. 20, 2012, which claims the benefit of U.S. provisional patent application Ser. No. 61/162,394, filed Mar. 23, 2009, entitled “METHODS FOR SIMULTANEOUS GENERATION OF FUNCTIONAL LIGANDS”, and U.S. Pat. No. 8,034,569, filed Jun. 6, 2009, entitled “METHODS FOR MOLECULAR DETECTION”, which claims the benefit of U.S. provisional patent application Ser. No. 61/059,435, filed Jun. 6, 2008, entitled “METHODS FOR MOLECULAR DETECTION”, the contents of all of which are hereby incorporated by reference in their entireties.
SEQUENCE LISTING
Deoxyribonucleic acid sequences are included in the ASCII text file entitled “PSEQ1_P1011US05.txt”, created May 28, 2013, of 96 kilobytes in size, which is hereby incorporated by reference in its entirety.


FIELD OF THE INVENTION
This invention relates to functional ligands to target molecules, particularly to functional nucleic acids and modifications thereof and methods for generating functional ligands, particularly to methods for generating multiple functional nucleic acids against multiple different target molecules simultaneously.
BACKGROUND OF THE INVENTION
Aptamers, which are nucleic acid ligands capable of binding to molecular targets, have recently attracted increased attention for their potential application in many areas of biology and biotechnology. They may be used as sensors, therapeutic tools, to regulate cellular processes, as well as to guide drugs to their specific cellular target(s). Contrary to the actual genetic material, their specificity and characteristics are not directly determined by their primary sequence, but instead by their tertiary structure. Aptamers have been recently investigated as immobilized capture elements in a microarray format. Others have recently selected aptamers against whole cells and complex biological mixtures.
Aptamers are commonly identified by an in vitro method of selection sometimes referred to as Systematic Evolution of Ligands by EXponential enrichment or “SELEX”. SELEX typically begins with a very large pool of randomized polynucleotides which is generally narrowed to one aptamer ligand per molecular target. Once multiple rounds (typically 10-15) of SELEX are completed, the nucleic acid sequences are identified by conventional cloning and sequencing. Aptamers have most famously been developed as ligands to important proteins, rivaling antibodies in both affinity and specificity, and the first aptamer-based therapeutics are now emerging. More recently, however, aptamers have been also developed to bind small organic molecules and cellular toxins, viruses, and even targets as small as heavy metal ions.
SUMMARY OF THE INVENTION
The present invention relates functional ligands to target molecules, particularly to functional nucleic acids and modifications thereof, and to methods for simultaneously generating, for example, numerous different functional biomolecules, particularly to methods for generating numerous different functional nucleic acids against multiple target molecules simultaneously. The present invention further relates to functional ligands which bind with affinity to target molecules. The present invention further relates to methods for generating, for example, functional biomolecules, particularly to functional nucleic acids, that bind with functional activity to another biomolecule, such as a receptor molecule. More than one or multiple targets as used herein may generally include different types of targets, and/or may also include a multitude of a singular type of targets at different conditions, such as, for example, temperature, pH, chemical environment, and/or any other appropriate conditions.
In general, a method for generating functional biomolecules includes obtaining a library, such as a diverse or randomized library, for example, of biomolecules. Biomolecules may generally include nucleic acids, particularly single-stranded nucleic acids, peptides, other biopolymers and/or combinations or modifications thereof. A library of biomolecules may include nucleic acid sequences, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/or combinations thereof. The method for generating functional biomolecules further includes contacting the library of biomolecules with more than one target, such as, for example, a molecular target, material and/or substance. In general, the members of the library that do not bind with some affinity to the more than one target may be washed or otherwise partitioned from the remainder of the library, which may have a given level of binding affinity to the more than one target. The process may be repeated to partition the strongest binding members of the library. Amplification of the biomolecules may also be utilized to increase the numbers of the binding members of the library for subsequent repetitions and for isolation and/or purification of any final products of the process. Embodiments of the SELEX method may generally be utilized to achieve the generation of functional biomolecules of a given binding affinity, such biomolecules generally referred to as aptamers or ligands.
In one exemplary aspect of the invention, generation of functional biomolecules may be performed against more than one or multiple targets simultaneously within a single system, such as the generation of functional nucleic acid ligands within a single reaction volume. In general, more than one or a plurality of targets may be disposed within in a single reaction volume, and a library of biomolecules, such as a nucleic acid library, may be applied to the reaction volume. The members of the library that do not bind to any of the plurality of targets under given conditions may then be partitioned, such as by washing. One or more rounds of binding and partitioning of the members of the library may be performed, such as, for example, to obtain a remainder of members of the library with a given affinity for their targets. The remaining members that bind to the plurality of targets of the library may then be marked and/or tagged, such as to identify the particular target or targets to which the member(s) of the library binds. The binding members of the library may then be isolated and, by virtue of the marking or tagging, be matched to a particular target or targets. This is desirable as high capacity, multiplexed identification procedures may save time, expense, and physical space for the process over single target identification processes. The present method may also be desirable as it may be utilized to identify and/or eliminate biomolecules that bind or have a tendency to bind to multiple targets.
In an exemplary embodiment, a plurality of target molecules are affixed to a substrate within a single reaction volume, such as, for example, by attaching the targets to a substrate of an array. It may generally be appreciated that a single reaction volume may refer to or include multiple reaction sub-volumes, such as, for example, discrete or semi-discrete fluid droplets. In general, the targets may be disposed with multiple copies of each target in clusters or “spots” such that a given array may have an ordered deposition of targets on the substrate, with each target identifiable by the location of a particular spot on the substrate. A library of nucleic acids may then be contacted or applied to the array and the non-binding members of the library may be partitioned or washed off the array. The binding and washing steps may be repeated and may also utilize an amplification step to generate additional copies of any remaining binding members of the library. The array may then be marked or tagged with a plurality of identifiers, such as, for example, a plurality of oligonucleotides which may universally bind through Watson-Crick interactions to the members of the library of nucleic acids. The marking or tagging may be, for example, accomplished by manually applying identifiers, such as by pipetting or the like, utilizing microcontact pins, applying membranes/films with identifiers, printing, for example, inkjet printing, and/or other similar tagging methods, of identifier containing solutions to the array. The identifiers may further include a unique or semi-unique sequence which may be utilized to correspond to the spots and thus the targets of the array. For example, a unique or semi-unique identifier sequence may be utilized that identifies each spatial location on an array, such as each particular target spot. The identifier may then be associated with and/or attached to the nucleic acid members bound to a particular spot. Thus, the nucleic acids, for example, bound to a particular target spot may be identified by the sequence of the associated identifier. In some embodiments, the identifiers may further be primers and may be utilized with a nucleic acid amplification reaction on the array to generate additional copies of the bound nucleic acids. The unique or semi-unique identifier sequence may also be incorporated into the members of the library amplified. This may be desirable for associating a given member with a target or targets while preserving the particular sequence of the member as the locational identifying sequence is appended to the sequence of the library member. This may be particularly desirable for resolving multiple binders to a single target or members of the library that bind to multiple targets.
In general, the starting library of biomolecules, such as nucleic acids, may be the product of at least one round of a previous SELEX protocol. For example, at least one round of SELEX may be performed with a library of biomolecules against multiple targets, such as, for example, in a solution. The targets in the solution may be substantially identical to the targets disposed on an array. This may be desirable as multiple rounds of selection may be performed with a library prior to applying the remaining members to an array for marking/tagging. Complex target arrays may generally be more expensive and/or difficult to make or utilize than solutions of target molecules, so performing only the final binding and marking/tagging procedure on the array may be desirable.
In other embodiments, identifiers may be predisposed on the array substrate in substantial proximity to the spots such that they may bind to, for example, nucleic acids bound to the target spots. The identifiers may, for example, be covalently attached to the substrate. In some embodiments, the attachments may be controllably breakable or cleavable such that the identifiers may be released from the substrate such that they may, for example, more easily bind to the bound nucleic acids on the spots.
In further embodiments, identifiers may be synthesized in situ on the array, such as by light directed in situ nucleic acid synthesis. Appropriately sequenced identifiers may then be synthesized in proximity to particular spots such that the newly synthesized identifiers may bind to the nucleic acids bound to the target spot.
In still other embodiments, identifiers may be disposed and/or synthesized on a separate substrate, such as a membrane, in a spatial disposition that substantially matches the spatial disposition of spots on the array, i.e. the identifiers may be arranged such that they may be readily superimposed onto the target spots on the array. The identifier substrate may then be contacted with the array with locational matching of the spots with identifiers. The identifiers may then bind to the nucleic acids bound to the target spots. Any appropriate method of facilitating binding may be utilized, such as, for example, actions to drive migration of the identifiers to the array, such as capillary action, electrophoresis, pressure, gravitational settling, and/or any other appropriate method or combination thereof. The separate substrate may also be soluble, erodible, substantially permeable to the identifiers, and/or otherwise adapted for facilitating migration of the identifiers to the array.
In yet still other embodiments, the array substrate may be physically divided and/or partitioned for separate collection of the, for example, nucleic acids bound to the spots. The spots may, for example, also be controllably removable from the substrate such that they may be individually recovered and sorted.
In still yet other embodiments, identifiers may be disposed and/or synthesized on a separate substrate, such as a membrane, in a spatial disposition that substantially matches the spatial disposition of spots on the array, i.e. the identifiers may be arranged such that may be readily superimposed onto the target spots on the array. The separate substrate may be kept separately while the array substrate maybe physically divided and/or partitioned for separate collection of the nucleic acids bound to the spots. In this manner, the location of the different nucleic acids maybe maintained even when the array substrate is no longer intact, if the locations are of value. The identifiers may also be selectively applied to particular locations on the array and/or applied in a particular order or in groups.
In some embodiments, identifiers may only be applied to spots with bound nucleic acids. The spots with bound nucleic acids may be detected, for example, by detecting the presence of nucleic acids, such as by applying nucleic acid binding dyes, such as SYBR dyes, ethidium bromide and/or other appropriate dyes. The members of the nucleic acid library may also include detectable portions, such as, for example, fluorescent moieties, radioactive tags and/or other appropriate detectable portions.
In some embodiments, the identifiers may be applied to the bound nucleic acids together with other materials, such as for example, components of a nucleic acid amplification reaction, a nucleic acid ligation reaction, photo-linking reagents, and/or any other appropriate material, such as those materials that may facilitate attachment or association of the identifiers to the bound nucleic acids.
In yet another embodiment, identifiers may be ligated to the, for example, bound nucleic acids. For example, a nucleic acid ligase may be utilized to covalently link an identifier sequence to the bound nucleic acid. Further nucleic acid fragments may be utilized to facilitate ligase action, such as appropriate complementary fragments that may aid the formation of a substantially double-stranded nucleic acid complex compatible with a ligase. For another example, photo-ligation may be used to attach the identifiers to the, for example, bound nucleic acids. Photo-ligation may be especially useful when certain substrates are used. For example, macro-porous substrates.
In general, methods may be applied that may facilitate binding or other interactions between the identifiers and the, for example, nucleic acids bound to the spots. For example, the temperature may be increased to dissociate the nucleic acids from the spots. The temperature may subsequently be lowered such that, for example, base pairing may occur between the nucleic acids and the identifiers. Further in general, it may be desirable to apply the identifiers in a manner that physically separates and/or isolates the individual target spots such that cross-marking due to identifier diffusion/migration may be minimized. For example, the identifiers may be applied in individual fluid droplets such that there is no continuous fluid contact between individual identifier containing fluids. For further example, the substrate of the array may be absorbent and/or porous such that the identifiers may be absorbed into the substrate material. The substrate material may also block lateral diffusion while allowing vertical diffusion, such that identifiers may be applied and absorbed into the substrate while minimizing diffusion across the plane of the substrate, such as to other target spots.
In a further embodiment, a method for generating functional biomolecules includes obtaining a library of peptide sequences and contacting the library with a plurality of targets. In some exemplary embodiments, the peptide sequence may be tagged, linked, marked and/or otherwise associated with a nucleic acid sequence. The nucleic acid sequence may be, for example, representative of the sequence of the peptide. For example, the nucleic acid may substantially encode the peptide sequence. Also for example, the nucleic acid may be a unique or semi-unique identifier sequence. The nucleic acid sequence may then be utilized to bind another identifier, as described above, such that a peptide bound to a target may be tagged or marked as to which target it bound.
In an exemplary embodiment, a bacteriophage (phage) may be generated that includes a peptide sequence of interest in its protein coat. The phage may further include a nucleic acid sequence that may be representative of the peptide sequence within the nucleic acid of the phage. The phage may then be contacted with a plurality of targets, as above. This may generally be referred to as phage display. Non-binding phages may be washed and/or partitioned, while binding phages may be tagged or marked with identifiers, as above. As phage nucleic acids are generally contained within the protein coat of the phage, the nucleic acid may generally be exposed for binding to the identifier. For example, the phage may be heated such that the protein coat denatures and/or disassembles such that the nucleic acid is exposed. The identifier may also be introduced into the phage, such as by electroporation, electrophoresis, and/or any other appropriate method.
Other methods of peptide selection may include, but are not limited to, mRNA display, ribosome display, and/or any other appropriate peptide display method or combination thereof.
In another aspect of the invention, methods for handling and sorting the resultant sequences of a multiplexed binding process are provided. In some embodiments, the sequences may be sorted by identifier sequences to establish which target or targets the sequence bound. The sequences may further be compared, aligned and/or otherwise processed to identify features, characteristics and/or other useful properties, relationships to each other, and/or target properties.
In a further aspect of the invention, methods for monitoring and/or controlling the diversity of the library of biomolecules may be utilized. For example, too few rounds of selection may result in a biomolecule pool with too many weak binding members while too many rounds of selection may result in only a few binding members, such as members corresponding to only a few targets rather than members corresponding to all of the targets present. In one embodiment, Cot analysis may be employed to measure and/or monitor the diversity of the library of biomolecules through multiple rounds of selection. Cot, or Concentration×time, analysis measures the annealing time of particular oligonucleotides while in solution with other nucleic acids, such as the members of the library of biomolecules. In general, the annealing time will be faster the lower the diversity of the library.
The present invention together with the above and other advantages may best be understood from the following detailed description of the embodiments of the invention illustrated in the drawings.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates an embodiment of a multiple target array;
FIG. 2 illustrates the application of a library of biomolecules to a target array;
FIG. 2a illustrates the binding of members of a library of biomolecules to a target spot;
FIGS. 3 and 3a illustrate embodiments of biomolecule library members;
FIG. 3b illustrates an embodiment of an identifier;
FIG. 4 illustrates the tagging of a library member bound to target with an identifier;
FIG. 4a illustrates a tagged library member product;
FIG. 5 illustrates a target spot with nearby identifiers on a substrate;
FIG. 5a illustrates the application of an identifier sheet to a target array;
FIGS. 6, 6a, 6b and 6c illustrate embodiments of identifiers and ligation of identifiers to a library member;
FIGS. 7 and 7a illustrate phage display for a target;
FIG. 7b illustrates an mRNA display fusion product;
FIG. 7c illustrates a ribosome display fusion product; and
FIG. 8 illustrates an example of a histology section target.



DETAILED DESCRIPTION OF THE INVENTION
The detailed description set forth below is intended as a description of the presently exemplified methods, devices, and compositions provided in accordance with aspects of the present invention and is not intended to represent the only forms in which the present invention may be practiced or utilized. It is to be understood, however, that the same or equivalent functions and components may be accomplished by different embodiments that are also intended to be encompassed within the spirit and scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the exemplified methods, devices and materials are now described.
The present invention relates functional ligands to target molecules, particularly to functional nucleic acids and modifications thereof, and to methods for simultaneously generating, for example, numerous different functional biomolecules, particularly to methods for generating numerous different functional nucleic acids against multiple target molecules simultaneously. The present invention further relates to functional ligands which bind with affinity to target molecules. The present invention further relates to methods for simultaneously generating different functional biomolecules, particularly to functional nucleic acids, that bind with functional activity to another biomolecule, such as a receptor molecule.
In general, a method for generating functional biomolecules includes obtaining a library, such as a diverse or randomized library, of biomolecules. Biomolecules may generally include nucleic acids, particularly single-stranded nucleic acids, peptides, other biopolymers and/or combinations or modifications thereof. A library of biomolecules may include nucleic acid sequences, such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), artificially modified nucleic acids, and/or combinations thereof. In general, modified nucleic acid bases may be utilized and may include, but are not limited to, 2′-Deoxy-P-nucleoside-5′-Triphosphate, 2′-Deoxyinosine-5′-Triphosphate, 2′-Deoxypseudouridine-5′-Triphosphate, 2′-Deoxyuridine-5′-Triphosphate, 2′-Deoxyzebularine-5′-Triphosphate, 2-Amino-2′-deoxyadenosine-5′-Triphosphate, 2-Amino-6-chloropurine-2′-deoxyriboside-5′-Triphosphate, 2-Aminopurine-2′-deoxyribose-5′-Triphosphate, 2-Thio-2′-deoxycytidine-5′-Triphosphate, 2-Thiothymidine-5′-Triphosphate, 2′-Deoxy-L-adenosine-5′-Triphosphate, 2′-Deoxy-L-cytidine-5′-Triphosphate, 2′-Deoxy-L-guanosine-5′-Triphosphate, 2′-Deoxy-L-thymidine-5′-Triphosphate, 4-Thiothymidine-5′-Triphosphate, 5-Aminoallyl-2′-deoxycytidine-5′-Triphosphate, 5-Aminoallyl-2′-deoxyuridine-5′-Triphosphate, 5-Bromo-2′-deoxycytidine-5′-Triphosphate, 5-Bromo-2′-deoxyuridine-5′-Triphosphate, 5-Fluoro-2′-deoxyuridine-5′-Triphosphate, and/or any other appropriate modified nucleic acid base. It may generally be understood that the nucleoside triphosphates (NTPs) listed above may generally refer to any appropriate phosphate of the modified base, such as additionally, for example, monophosphates (NMPs) or diphosphates (NDPs) of the base. The method for generating functional biomolecules further includes contacting the library of biomolecules with at least one target, such as, for example, a molecular target, material and/or substance. In general, the members of the library that do not bind with some affinity to the target may be washed or otherwise partitioned from the remainder of the library, which may have a given level of binding affinity to the target. The process may be repeated to partition the strongest binding members of the library. Amplification of the biomolecules may also be utilized to increase the numbers of the binding members of the library for subsequent repetitions and for isolation and/or purification of any final products of the process. Embodiments of the SELEX method may generally be utilized to achieve the generation of functional biomolecules of a given binding affinity. The basic SELEX protocol and aptamers are described in U.S. Pat. No. 5,270,163, entitled “Methods for identifying nucleic acid ligands,” the entire contents of which are hereby incorporated by reference.
In one exemplary aspect of the invention, generation of functional biomolecules may be performed against multiple targets simultaneously within a single system, such as the generation of functional nucleic acid ligands within a single reaction volume. In general, a plurality of targets may be disposed within in a single reaction volume and a library of biomolecules, such as a nucleic acid library, may be applied to the reaction volume. The targets may be, for example, proteins, cells, small molecules, biomolecules, and/or combinations or portions thereof. The members of the library that do not bind to any of the plurality of targets under given conditions may then be partitioned, such as by washing. The remaining members of the library may then be marked and/or tagged, such as to identify the particular target or targets to which the member of the library binds. The binding members of the library may then be isolated and, by virtue of the marking or tagging, be matched to a particular target or targets. This may be desirable as high capacity, multiplexed identification procedures may save time, expense, and physical space for the process over single target identification processes. The present method may also be desirable as it may be utilized to identify and/or eliminate molecules that bind to multiple targets.
In an exemplary embodiment, a plurality of target molecules are affixed to a substrate within a single reaction volume, such as, for example, by attaching the targets to a substrate of an array. As illustrated in FIG. 1, the targets may be disposed with multiple copies of each target, such as target molecules, in clusters or “spots” 110 on the substrate 102 of an array 100 such that a given array 100 may have an ordered deposition of targets on the substrate 102, with each target identifiable by the location of a particular spot 110 on the substrate 102. Each spot 110 may be a unique target or there may be multiple spots 110 of at least one target on a given array 100. In general, high content target arrays, such as high content protein or antibody arrays, may be utilized. A library 200 of, for example, nucleic acids 202 may then be applied A to array 100, as illustrated in FIG. 2. Particular members 204 of the library 200 may then bind to target spots 110, such as illustrated in FIG. 2a. The non-binding members 206 of the library 200 may be partitioned or washed off the array 100. The binding and washing steps may be repeated and may also utilize an amplification step to generate additional copies of any remaining binding members 204 of the library 200. The array 100 may then be marked or tagged with a plurality of identifiers, such as, for example, a plurality of oligonucleotides which may universally bind through Watson-Crick interactions to the members of the library of, for example, nucleic acids. In one embodiment, each member 202 of the library 200 may include a potential binding sequence 202a and at least one conserved region 202b which may bind an identifier oligonucleotide, such as illustrated in FIG. 3. A further conserved region 202c may also be included to facilitate priming for amplification reactions, such as Polymerase Chain Reaction (PCR), as illustrated in FIG. 3a. In general the conserved regions 202b, 202c may flank the potential binding sequence 202a, such as to facilitate priming for amplification. An identifier 302 may then include a unique or semi-unique sequence 302a, such as illustrated in FIG. 3b, which may be utilized to correspond to the spots 110 and thus the targets of the array 100 by location of the a spot 110 on the substrate 102. The identifiers 302 may further include conserved region 302b which may bind to the conserved region 202b of the library members 202 by Watson-Crick base pairing. The identifiers 302 may also include a further conserved region 302c which may facilitate priming for amplification. The identifiers 302 may be, for example, applied to the spots 110 by printing, for example, inkjet printing, using micro-contact pins, and/or otherwise applying solutions containing identifiers 302 to the substrate 102 of the array 100, such as, for example, by pipetting or the like, onto the spots 110. A library member 202 bound to a target spot 110 may then be tagged with an identifier 302 via base pairing B at regions 202b, 302b, as illustrated in FIG. 4. Thus, the nucleic acids 202 bound to a particular target spot 110 may be identified by the sequence 302a of the identifier 302. In an exemplary embodiment, nucleic acid amplification, such as PCR, may be utilized to generate copies of the members 202 bound to the spots 110, incorporating the identifier sequence 302a (or more its complementary sequence) into the product 203, as illustrated in FIG. 4a. This may be desirable for associating a given member 202 with a target or targets 110 while preserving the particular sequence of the member 202. This may be particularly desirable for resolving multiple binders to a single target or members of the library that bind to multiple targets. Subsequent amplifications may utilize primers for the sequences 202c, 302c such that only the products 203 containing both the sequences 202a, 302a are amplified. It may be understood that references to nucleic acid sequences, such as above, may generally refer to either a particular sequence or the corresponding complementary nucleic acid sequence. In general, it may be desirable for single droplets and/or otherwise separated volumes of solutions containing identifiers 302 for each spot 110 on the array 100 such that the possibility of mistagging may be reduced.
In one aspect, the identifiers may be printed on all the targets. In another aspect, the identifiers may be printed only on targets with bound biomolecules.
In another embodiment, a histology section, such as the section 110′′ on substrate 102′′ of histology slide 100′′ in FIG. 8, may be utilized as a target set. The section 110′′ may be, for example, a tissue section, a cell mass, and/or any other appropriate biological sample which may generally have structurally significant features. As with the array 100, a library of biomolecules, such as nucleic acids, may be applied which may bind to specific locations on the section 110′′, the locations on which may, for example, represent separate targets to generate affinity binding nucleic acids. Identifiers may then be disposed on the slide 100′′ as described above, or as in the embodiments below, such that identifiers may be utilized to correspond to specific features of the section 110′′.
In other embodiments, identifiers may be predisposed on the array substrate in substantial proximity to the spots, such as illustrated with identifiers 302 disposed on substrate 102 in proximity to spot 110 in FIG. 5, such that they may bind to nucleic acids bound to the target spots. The identifiers may, for example, be covalently attached to the substrate. In some embodiments, the attachments may be controllably breakable or cleavable such that the identifiers may be released from the substrate such that they may, for example, more easily bind to the bound nucleic acids on the spots.
In further embodiments, identifiers may be synthesized in situ on the array, such as by light directed in situ nucleic acid synthesis. Appropriately sequenced identifiers may then be synthesized in proximity to particular spots such that the newly synthesized identifiers may bind to the nucleic acids bound to the spot.
In still other embodiments, identifiers may be disposed and/or synthesized on a separate substrate, such as a membrane, in a spatial disposition that matches the spatial disposition of spots on the array. FIG. 5a illustrates an example of an identifier sheet 100′ with membrane 102′ which may include identifier spots 110′ which may substantially correspond to target spots 110 of the array 100. The identifier sheet 100′ may then be contacted C with the array 100 with locational matching of the target spots 110 with identifier spots 110′. The identifiers may then bind to the nucleic acids bound to the target spots. Any appropriate method of facilitating binding may be utilized, such as, for example, actions to drive migration of the identifiers to the array, such as capillary action, electrophoresis, pressure, gravitational settling, and/or any other appropriate method or combination thereof.
In some embodiments, the membrane may be soluble and/or substantially erodible. For example, the membrane may include a film forming and/or soluble material. Identifiers and/or other materials, such as components of a nucleic acid amplification or ligation reaction, may be included such that a film is formed containing the desired materials. The membrane may then be applied to the substrate and a suitable solvent, such as water or ethanol, may be utilized to dissolve and/or erode the film, which may then release the included materials, such as the identifiers, to the substrate. Suitable materials for the film may include hydrophilic materials including polysaccharides such as carrageenan, chondroitin sulfate, glucosamine, pullulan, soluble cellulose derivatives such as hydroxypropyl cellulose and hydroxymethyl cellulose, polyacrylic acid, polyvinyl alcohol, polyethylene glycol (PEG), polyethylene oxide (PEO), ethylene oxide-propylene oxide co-polymer, polyvinylpyrrolidone (PVP), polycaprolactone, polyorthoesters, polyphosphazene, polyvinyl acetate, and polyisobutylene.
The membrane may further be adapted to have a desirable rate of erosion and/or dissolution. The rate may be modified by the inclusion of hydrophobic and/or less soluble additives. Suitable materials may include, but are not limited to, those from the family of quaternary ammonium acrylate/methacrylate co-polymers, (Eudragit RS), cellulose and its lower solubility derivatives, such as butyl cellulose, hydroxybutyl cellulose and ethylhydroxyethyl cellulose, high molecular weight PEG or PEO or a combination thereof.
In yet still other embodiments, the array substrate may be physically divided and/or partitioned for separate collection of the nucleic acids bound to the spots. The spots may, for example, also be controllably removable from the substrate such that they may be individually recovered and sorted. The array itself may also be perforated and/or otherwise easily and/or conveniently partitionable.
In another embodiment, identifiers may be ligated to the bound nucleic acids. For example, a nucleic acid ligase may be utilized to covalently link an identifier sequence to the bound nucleic acid. In general, nucleic acid ligases are enzymes that covalently join two nucleic acids by catalyzing the formation of phosphodiester bonds at the ends of the phosphate backbone of the nucleic acids. Examples of appropriate nucleic acid ligases may include, but are not limited to, E. coli DNA ligase, T4 DNA ligase, T4 RNA ligase, strand break DNA repair enzymes, and/or any other appropriate ligase, modified enzyme, and/or a combination thereof. In general the ligase utilized may be selected based on the form of ligation performed, such as ligation of blunt ends, compatible overhang (“sticky”) ends, single stranded DNA, singe stranded RNA and/or any other form of ligation. Further in general, the steps in ligating two nucleic acids together is a one step process that may be carried out at or near room temperature. Further nucleic acid fragments may be utilized to facilitate ligase action, such as appropriate complementary fragments that may aid the formation of a substantially double-stranded nucleic acid complex compatible with a ligase. In general, double stranded ligation may be employed and may utilize substantially compatible overhang fragments to facilitate ligation, or also blunt end ligation may be utilized, such as with either the nucleic acid end or the identifier having a phosphorylated end while the other is unphosphorylated for ligation. Single stranded ligation may also be employed.
Photo ligation may also be employed. Photo ligation may, for example, include covalently linking adjacent nucleic acids by application of electromagnetic energy, such as ultraviolet or visible light. Coupling agents may also be utilized to facilitate the formation of covalent linkages.
In some embodiments, dyes may be included into the identifiers. In one aspect, the identifiers may be doped with dyes. In another aspect, the identifier solutions may be mixed with dyes. According to one embodiment, the dyes may be photosensitive and may be fluorescent. According to another embodiment, the dyes maybe photosensitive and may be phosphorescent.
The substrates used may be glass, ceramic or polymeric, as long as their surfaces promote adhesion between the substrates and the targets. Polymers may include synthetic polymers as well as purified biological polymers. The substrate may also be any film, which may be non-porous or macroporous.
The substrate may be generally planar and may be of any appropriate geometry such as, for example, rectangular, square, circular, elliptical, triangular, other polygonal shape, irregular and/or any other appropriate geometry. The substrate may also be of other forms, such as cylindrical, spherical, irregular and/or any other appropriate form.
Appropriate ceramics may include, for example, hydroxyapatite, alumina, graphite and pyrolytic carbon.
Appropriate synthetic materials may include polymers such as polyamides (e.g., nylon), polyesters, polystyrenes, polyacrylates, vinyl polymers (e.g., polyethylene, polytetrafluoroethylene, polypropylene and polyvinyl chloride), polycarbonates, polyurethanes, poly dimethyl siloxanes, cellulose acetates, polymethyl methacrylates, ethylene vinyl acetates, polysulfones, nitrocelluloses and similar copolymers. These synthetic polymers may be woven or knitted into a mesh to form a matrix or similar structure. Alternatively, the synthetic polymer materials can be molded or cast into appropriate forms.
Biological polymers may be naturally occurring or produced in vitro by fermentation and the like or by recombinant genetic engineering. Recombinant DNA technology can be used to engineer virtually any polypeptide sequence and then amplify and express the protein in either bacterial or mammalian cells. Purified biological polymers can be appropriately formed into a substrate by techniques such as weaving, knitting, casting, molding, extrusion, cellular alignment and magnetic alignment. Suitable biological polymers include, without limitation, collagen, elastin, silk, keratin, gelatin, polyamino acids, polysaccharides (e.g., cellulose and starch) and copolymers thereof.
Any suitable substrate may be susceptible to adhesion, attachment or adsorption by targets. The susceptibility may be inherent or modified. In one example, the surfaces of substrates may be susceptible to adhesion, attachment or adsorption to proteins. In another example, the surfaces of substrates may be susceptible to adhesion, attachment or adsorption to proteins and not to nucleic acids.
In one exemplary embodiment, a glass substrate may have a layer or coating of a material that promotes adhesion with targets, such as proteins, materials that maybe charged, such as those that are positively charged, for binding target materials. Examples of charged materials include cellulosic materials, for example, nitrocellulose, methylcelluose, ethylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, methylhydroxypropyl cellulose; epoxies, PVDF (polyvinylidene fluoride); partially or fully hydrolyzed poly(vinyl alcohol); poly(vinylpyrrolidone); poly(ethyloxazoline); poly(ethylene oxide)-co-poly(propylene oxide) block copolymers; polyamines; polyacrylamide; hydroxypropylmethacrylate; polysucrose; hyaluronic acid; alginate; chitosan; dextran; gelatin and mixtures and copolymers thereof.
In another exemplary embodiment, if the substrate is not susceptible for attachment by charged materials, or may be susceptible only for attachment by wrongly charged materials, some areas of the substrate may have adhesives, binding agents, or similar attached, adsorbed or coated thereon. Examples of adhesives may include any suitable adhesives that bind the charged materials.
The targets may be present on the substrate discretely or in clusters. The distance between the discrete targets may be close or may be far apart and may usually be of different targets. Clusters may be used for multiple spots of a single target.
In one embodiment, the substrate may be macroporous. Macroporous substrates may be desirable, for example, if the different targets are very close together. When the targets are close by, there may not be sufficient distance between different targets to distinguish which target a biomolecule may be binding to. Closely packed targets may increase the efficiency of the generating of biomolecules. A macroporous substrate may be suited for balancing between efficiency and separation. For a macroporous substrate, the walls of the pores may be sufficient to separate even closely packed targets if the pores are large enough to enable the binding process to occur within the pores.
Also, for macroporous substrates, the pores may have an average diameter greater than the average size of the target material such that the target material may enter or partly enter the pores to anchor. Hydrogels may also be useful for binding or anchoring targets to the pores. Hydrogels may also fill the pores under fluid conditions and present a smooth surface for fluid flow while at the same time may keep the fluid from flowing through the pores.
The plurality of targets may be arranged in any appropriate manner such as, for example, in circular or elliptical spots, square or rectangular spots, stripes, concentric rings and/or any other appropriate arrangement on the subject.
According to one exemplary embodiment, the substrate may be at ambient temperature throughout.
According to another exemplary embodiment, the substrate may include a temperature affecting system that generally produces at least one desired temperature on the surface of the substrate and the adjacent fluid. The desired temperature may facilitate the biomolecule generating process.
According to a further exemplary embodiment, the substrate may include a temperature affecting system for producing a range of desired temperatures on the surface of the substrate and the adjacent fluid. This may be particularly useful when employing a set of targets having a significant range of, for example Tms, or melting temperatures. In one embodiment, the system may include a plurality of temperature affecting devices that are in thermal communication with the substrate. The plurality of devices may generally be disposed such that they may each produce a desired temperature in a given locality on the surface of the substrate. The set of targets may also be distributed on the surface of the substrate such that the temperature at the location of a target is substantially at the Tm of the target.
Temperature affecting devices may be any appropriate device that may substantially produce a desired temperature on a substrate and may include, but are not limited to, thermoelectric devices such as Peltier junction devices, semiconductor heating devices, resistive heating devices, inductive heating devices, heating/cooling pumps, electromagnetic radiation sources and/or any other appropriate devices. Temperature may also be affected by other systems, such as, for example, fluid flows including, but not limited to, water flows, air flows, and/or any other appropriate fluid flows.
In an exemplary embodiment, a plurality of Peltier junction devices may be utilized to generate desired temperatures at localities on the surface of the substrate. Peltier junction devices are particularly useful since they are able to both heat and cool using electrical current. This enables Peltier junction devices to generate temperatures above and below the ambient temperature of a system. They may also be useful in maintaining given temperature conditions at a steady state by adding and removing heat as necessary from the system.
In general, the placement of the temperature affecting devices may determine the temperature profile on the surface of the substrate and the adjacent fluid in the chamber. The temperature affecting devices may thus be disposed at appropriate positions such that given temperatures may be produced and maintained at known positions on the substrate.
The substrate may in general have a given thermal conductivity such that the application of at least one temperature affecting device may substantially generate a temperature gradient profile on the surface of the substrate. In general, the temperature on the surface of the substrate may change as a function of the distance from the position of the at least one temperature affecting device. Substrate materials with a relatively low thermal conductivity may generally produce highly localized temperature variations around a temperature affecting device. Substrate materials with a relatively high thermal conductivity may generally produce more gradual variations in temperature over a given distance from a temperature affecting device. It may be understood that at steady state, the effect of the thermal conductivity of the substrate may not contribute to the temperature profile of the system.
In some embodiments, at least one temperature affecting device may be utilized to produce a particular temperature gradient profile on the surface of the substrate. In general, a temperature gradient may be generated by utilizing at least one temperature affecting device producing a temperature different from the ambient temperature of the system. Multiple temperature affecting devices with at least two producing different temperatures may be utilized to generate a temperature gradient without reliance on the ambient temperature of the system.
The positions and temperatures of multiple temperature affecting devices may be utilized to calculate a resulting temperature gradient profile on the surface of a substrate using standard heat transfer equations. An algorithm may then be utilized to calculate the optimal positions and/or temperatures for a plurality of temperature affecting devices to produce a desired temperature gradient profile on the surface of a substrate. The algorithm may be, for example, applied using a computational assisting system, such as a computer and or other calculatory device. This may be performed to tailor a temperature gradient profile to a particular substrate with a known disposition of targets of known and/or calculated Tm. Similarly, a set of targets of known and/or calculated Tm may be arranged on a substrate based on a temperature gradient profile. This may be desirable as placement of a target at a given location on a substrate may be accomplished more easily than tailoring a temperature profile to pre-existing locations of targets on a substrate. In general, a target may be disposed on the substrate at a temperature address within the temperature profile gradient. The temperature address may, for example, be substantially at the Tm of the target during operation of the molecular hybridization system, and/or any other appropriate temperature.
In another aspect, the molecular hybridization system includes an adjustable system for generating a temperature profile. The adjustable system generally includes a plurality of temperature affecting devices, each affecting the temperature at a particular location of a substrate.
Details of the temperature affecting systems may be found in, for example, U.S. utility patent application Ser. No. 12/249,525, filed on Oct. 10, 2008, entitled “METHODS AND DEVICES FOR MOLECULAR ASSOCIATION AND IMAGING”, the contents of all of which are hereby incorporated by reference.
FIG. 6 illustrates an example of an identifier sequence 302 and a complement identifier sequence 402. The complement sequence 402 may include a complement identifier region 402a which may be substantially complementary to identifier region 302a such that they may base pair bind. The complement sequence 402 may further include a primer region 402c which may also be complementary to primer region 302c of the identifier 302. Further, the complement sequence 402 may include a compatible end 402b which may be compatible with ligation to the end of another nucleic acid. As shown in FIG. 6a, a nucleic acid library member 202 may be bound to a spot 110. An identifier 302 and a complement sequence 402 may then be applied to the member 202 such that the identifier 302 binds to the member 202 at region 202b, 302b. The complement sequence 402 may bind to the identifier 302 at regions 302a/402a, 302c/402c. The compatible end 402b may then be ligated to the end D of the member 202 by an appropriate ligase and/or other appropriate method. A product 203′, as illustrated in FIG. 6b, may then be generated including the primer region 202c, binding sequence 202a, region 202b, complement identifier region 402a, and complement primer region 402c. The product 203′ may then be amplified, such as with the product 203 discussed above in FIG. 4a. The product 203′ may also be generated by single-stranded ligation of the member 202 and the complement sequence 402, where in general the either the member 202 or the complement sequence 402 may have a phosphorylated end while the other may be unphosphorylated for end to end ligation.
In another example, as illustrated in FIG. 6c, a further complementary fragment 502 may be included that may base pair bind to a complementary region 202d of the nucleic acid library member 202. This may be desirable as some nucleic acid ligases may generally join double stranded nucleic acids. The addition of the complementary fragment 502 may generally generate a substantially double stranded nucleic acid, such as illustrated spanning from region 302c to the end of complementary fragment 502. There may further be a double stranded “break” at points D and E. In general, the sizing of the fragments may be tailored to generate a suitably long stretch of double stranded nucleic acid for ligase action. In general, the complementary region 202d may be the same for all members 202 of the library 200 such that the same complementary fragment 502 may be utilized, such as, for example, convenience, cost and/or ease of use.
In general, methods may be applied that may facilitate binding or other interactions between the identifiers and the nucleic acids bound to the spots. For example, the temperature may be increased to dissociate the nucleic acids from the spots. The temperature may subsequently be lowered such that, for example, base pairing may occur between the nucleic acids and the identifiers. Temperature changes may also, for example, denature the target such that the nucleic acids may no longer bind and/or bind with lower affinity to the targets. This may be desirable in that it may aid in binding of the nucleic acids to the identifiers.
In a further aspect of the invention, methods for monitoring and/or controlling the diversity of the library of biomolecules may be utilized. For example, too few rounds of selection may result in a biomolecule pool with too many weak binding members while too many rounds of selection may result in only a few binding members, such as members corresponding to only a few targets rather than members corresponding to all of the targets present. In one embodiment, Cot analysis may be employed to measure and/or monitor the diversity of the library of biomolecules through multiple rounds of selection. Cot, or Concentration×time, analysis measures the annealing time of particular oligonucleotides while in solution with other nucleic acids, such as the members of the library of biomolecules. In general, the annealing time will be faster the lower the diversity of the library.
In one embodiment, a Cot-standard curve for measuring the sequence diversity of the aptamer library at any point during the multiplex SELEX process may be utilized. For example, a group of DNA oligonucleotides with a 5′- and 3′-constant region of  ̃20 bases identical to the initial SELEX library may be utilized. The oligos may then be converted to dsDNA by standard methods. Briefly, after annealing a primer to the oligos, Exo-minus Klenow Taq polymerase (Epicentre, Madison, Wis.) may be used in conjunction with dNTPs to fill in the ssDNA to create a dsDNA or mixture thereof. Using a standard quantitative PCR thermal cycler, a temperature profile for melting and controlled annealing of each DNA mixture may be programmed. Standard SYBR Green I specific for double-stranded DNA (dsDNA) may be utilized to report the amount of re-annealed dsDNA. At one extreme, the annealing time for a single sequence will be measured. At the other extreme, the annealing time for the initial SELEX pool, such as containing approximately 1 nmol of sequence diversity, may be measured. Annealing times of intermediate diversity may also be measured to establish a very specific Cot-standard-curve for the SELEX library. Using this standard curve, at any point during SELEX, the sequence diversity of the evolving library of aptamers may be determined by comparison to the curve.
In a further embodiment, a method for generating functional biomolecules includes obtaining a library of peptide sequences and contacting the library with a plurality of targets. In some embodiments, the peptide sequence may be tagged, linked, marked and/or otherwise associated with a nucleic acid sequence. The nucleic acid sequence may be, for example, representative of the sequence of the peptide. For example, the nucleic acid may substantially encode the peptide sequence. Also for example, the nucleic acid may be a unique or semi-unique identifier sequence. The nucleic acid sequence may then be utilized to bind another identifier, as described above, such that a peptide bound to a target may be tagged or marked as to which target it bound.
In an exemplary embodiment, a bacteriophage (phage) may be generated that includes a peptide sequence of interest in its protein coat. The phage may further include a nucleic acid sequence that may be representative of the peptide sequence within the nucleic acid of the phage. The phage may then be contacted with a plurality of targets, as above. This may generally be referred to as phage display. Phages employed may include, but are not limited to, M13 phage, fd filamentous phage, T4 phage, T7 phage, λ phage, and/or any other appropriate phage. Non-binding phages may be washed and/or partitioned, while binding phages may be tagged or marked with identifiers, as above. As phage nucleic acids are generally contained within the protein coat of the phage, the nucleic acid may generally be exposed for binding to the identifier. For example, the phage may be heated such that the protein coat denatures and/or disassembles such that the nucleic acid is exposed. The identifier may also be introduced into the phage, such as by electroporation, electrophoresis, and/or any other appropriate method.
In FIG. 7, an example of a phage 600 may include a nucleic acid 610 which may generally encode, among other things, and be encapsulated by a protein coat 602, which may contain a binding region for a target 110. The nucleic acid 610 may further include a region 612 which may identify the phage and/or encode the binding region for a target. A bound phage 600, as illustrated in FIGS. 7 and 7a, may then be heated, disrupted and/or otherwise treated such that an identifier 302 may contact F the region 612. For example, the protein coat 602 may be broken and/or otherwise disrupted for entry of the identifier 302. In general, an amplification reaction and/or other method, such as those discussed above, may be utilized to tag, mark and/or otherwise introduce identifier information to the sequence of region 612. Further in general, the identifier 302 and region 612 may incorporate any, all or a combination of the elements discussed above in regards to nucleic acid library members, identifiers and/or other nucleic acid fragments. As also discussed above, the phage 600 may also be physically removed and/or partitioned in a manner that may preserve the identity of the target 110 the phage 600 was associated.
In other embodiments, other methods of incorporating and/or linking nucleic acids to peptides may be utilized, such as, for example, mRNA display, ribosome display, and/or any other appropriate method. In general, in mRNA display, as illustrated in FIG. 7b, a fusion product 600′ of a messenger RNA (mRNA) 610′ may be linked to a peptide 602′ that the mRNA 610′ encodes, such as with a puromycin-ended mRNA 612′ which may generally cause fusion of the mRNA 610′ to the nascent peptide 602′ in a ribosome, which may then be contacted with targets such as described above with phage display. Also in general, in ribosome display, as illustrated in FIG. 7c, a fusion product 600′′ of a modified mRNA 610′′ may be utilized that codes for a peptide 602′′, but lacks a stop codon and may also incorporate a spacer sequence 612′′ which may occupy the channel of the ribosome 620′′ during translation and allow the peptide 602′′ assembled at the ribosome 620′′ to fold, which may result in the peptide 602′′ attached to the ribosome 620′′ and also attached to the mRNA 610′′. This product 600′′ may then be contacted with targets such as described above with phage display. Other methods may include, but are not limited to, yeast display, bacterial display, and/or any other appropriate method.
In another aspect of the invention, methods for handling and sorting the resultant sequences of a multiplexed binding process are provided. In some embodiments, the sequences may be sorted by identifier sequences to establish which target or targets the sequence bound. The sequences may further be compared, aligned and/or otherwise processed to identify features, characteristics and/or other useful properties, relationships to each other, and/or target properties. For example, it may be expected that multiple aptamer sequences bound to a single target may potentially share sequence motifs and/or other common features which may be at least partially elucidated by sequence sorting and/or comparison. Specific binding affinities of resultant sequences may also be determined through affinity assays. In some embodiments, surface plasmon resonance may be utilized to determine binding of an aptamer to a target. For example, sensors which monitor the refractive index of a surface bound to a target may be utilized, where the refractive index may change as a result of binding of an aptamer to the target. In general, aside from standard sequencing methods, parallel sequencing methods, such as, for example, massively parallel sequencing such as 454 Clonal Sequencing (Roche, Branford, Conn.), massively parallel clonal array sequencing, Solexa Sequencing (Illumina, San Diego, Calif.), and/or any other appropriate sequencing method may be employed.
Example of Multiplexed Selex Protocol
As a demonstration of parallel, de novo selection of aptamers against multiple targets, a combinatorial DNA library containing a core randomized sequence of 40 nts flanked by two nt conserved primer binding sites is used as the starting point for an aptamer pool. The primer sequences are designed and optimized using Vector NTI's (Invitrogen) oligo analysis module. Typically, such a library is expected to contain approximately 1015 unique sequences. The primer binding sites are used to amplify the core sequences during the SELEX process. The single stranded DNA pool dissolved in binding buffer is denatured by heating at 95° C. for 5 min, cooled on ice for 10 min and exposed to multiple protein targets fixed onto a nitrocellulose coated glass slide (e.g., Whatman).
Example of DNA Library Selex
An example DNA library consists of a random sequence of 40 nucleotides flanked by conserved primers. In the first round of SELEX, 500 pmol of the ssDNA pool is incubated with each slide in binding buffer (PBS with 0.1 mg/ml yeast tRNA and 1 mg/ml BSA) for 30 minutes at 37° C. The slide is then washed in 1 ml of binding buffer for one minute. To elute specifically bound aptamers the slide is heated to 95° C. in binding buffer. The eluted ssDNA is subsequently be precipitated using a high salt solution and ethanol. After precipitation, the aptamer pellet is resuspended in water and amplified by PCR with a 3′-biotin-labeled primer and a 5′-fluorescein (FITC)-labeled primer (20 cycles of 30 sec at 95° C., 30 sec at 52° C., and 30 sec at 72° C., followed by a 10 min extension at 72° C.). The selected FITC-labeled sense ssDNA is separated from the biotinylated antisense ssDNA by streptavidin-coated Sepharose beads (Promega, Madison, Wis.) for use in the next round. Alternatively, “asymmetric PCR” may be utilized for generating a large excess of an intended strand of a PCR product in SELEX procedures. Also alternatively, the undesired strand may be digested by λ-exonuclease, such as, for example, when a phosphorylated PCR primer is employed.
The labeling of individual aptamers with fluorescein isothiocyanate (FITC) facilitates the monitoring of the SELEX procedure. FITC is also compatible with scanning in the green (cy3) channel of standard microarray scanners. The sense primer used to amplify the ssDNA aptamers after each round of selection is fluorescently labeled, resulting in fluorescently labeled aptamers. The protein spotted nitrocellulose-coated slides are scanned in a microarray scanner. Alternatively, proteins may be spotted on epoxy-coated glass slides. While epoxy slides may have less protein binding capacity than 3-D nitrocellulose pads, it has been observed that there may be less non-specific binding of nucleic acid aptamer pools to the background of the slide (blocked or not). Blocking may be employed to reduce background fluorescence.
In each round of the SELEX process, the slide is incubated for 30 min at 37° C. to allow binding of the aptamers to their targets. The slides are then washed in binding buffer before the specifically bound DNAs are eluted by heating the slide at 95° C. in 7M urea. Nucleic acids from the eluate are phenol-chloroform purified and precipitated, and the concentrated single stranded DNA molecules will be amplified by PCR. In order to increase stringency throughout the SELEX process, the washes are gradually increased in volume (from approximately 1-10 ml). After a given point in the selection, such as, for example, after the final round of selection, the aptamers may be tagged, marked and/or partitioned.
Example of In Situ Hybridization of Identifiers
An example of in situ hybridization of identifiers to aptamers was performed with short, ssDNA sequence tags to the 3′ end of aptamers bound to their protein target. These synthetic ssDNA tag oligonucleotides consists of three regions, as illustrated in FIG. 3b with identifier 302: (i) the C2 region, region 302c of the identifier 302, at the 3′ end of the oligonucleotide consists of a 17-20 nucleotide sequence complementary to a corresponding region on all of the used aptamers, (ii) the C1 region, region 302b at the 5′ end of the oligonucleotide 302 contains a 17-20 nt primer binding site, used during the amplification of the tag:aptamer hybrid, prior to sequencing and (iii) a variable region 302a in the center of the tag oligonucleotide (V) that serves a as a unique identifier for each locus on the glass slide surface. A variable sequence of 8 nucleotides will allow 48 (65,536) unique sequences to be generated, sufficient for many complex protein arrays (8000 samples) on the market.
As outlined above, after the final round of the SELEX procedure (typically, round 10) the specific aptamers are bound to their protein targets, fixed to a glass slide. While the 40 nt core sequence of each aptamer are unique, its terminal sequences have not been subject to any kind of selection during the procedure. After each round of binding to their protein targets, the aptamers were amplified using conserved primers, requiring the maintenance of corresponding regions at their distal ends (P1, P2). The 3′-region of each aptamer, for instance, can thus serve as a binding site (via standard hybridization) for the C2 region of the proposed tag oligonucleotide. Given the unique variable sequence (V) of each tag oligonucleotide, each aptamer will now be tagged with a sequence that can be traced back to the location of the aptamer on the glass slide, and thus the protein spotted at that location.
Example of 16-Plex Selex Procedure
An example of a 16-plex (16 targets) SELEX procedure was performed with 16 unique targets: (1) fibrinogen, (2) collagen, (3) fibronectin, (4) acetyl-bovine serum albumin (BSA), (5) heparan sulfate, (6) prolactin inhibiting factor (PIF), (7) ribonuclease A (RNase A), (8) laminin, (9) interleukin-7 cat 200-7 (IL-7 cat 200-7), (10) IL-15 cat 200-15, (11) IL-21 cat 200-21, (12) IL-7R cat 306-IR, (13) IL-15R, (14) IL-21R, (15) IgG2a (anti-CD19), and (16) anti-CD20. The 16 targets were arranged as a spotted array on a blocked nitrocellulose slide. A DNA aptamer library as discussed above was applied to the slide. Non-binding members of the library were washed off and the slide was labeled with 16 unique identifier nucleic acid sequences, one per target spot, as described above. The identifiers were briefly incubated at 60° C. and then allowed to hybridize to the aptamers on the spots at 37° C. for 30 minutes. Exo-Klenow Taq polymerase and dNTPs were added and extension of the identifiers was performed for 1 hour at 37° C. The Taq was inactivated at 60° C. for 10 minutes. The dsDNA was eluted with near boiling 7M urea and then precipitated with 3M sodium acetate at pH 5.0 and ice cold ethanol. The recovered dsDNA was sequenced by standard methods.
Identifier sequences in the sequenced DNA were utilized to identify the target of the aptamer sequence. The following aptamer sequences were identified for the targets (identifier sequences and priming sequences removed):







Target 1:


(SEQ ID NO.: 1)


5′-CAAGAGTGTTAGACATTATCTCAGCGCTGCCAATTATATT-3′


 


Target 2:


(SEQ ID NO.: 2)


5′-AGAGGCGGCTGAGATCAATCTCCGCTCAGGGAGCGAGTA-3′


 


Target 3:


(SEQ ID NO.: 3)


5′-CAATACAATACTATATTTGTGTCAATCTCGTACTTCTGAC-3′


 


(SEQ ID NO.: 4)


5′-CAATATGTCTAATTTTTTTACATGGCGGCATGGTATTGGC-3′


 


Target 4:


(SEQ ID NO.: 5)


5′-AAGATCTTTATTAAGCAAACAATGTTAACTATAGAGCGTT-3′


 


(SEQ ID NO.: 6)


5′-GAATTACATTCAAAAATTTTCTTCTGGCATCTGTAATACCG-3′


 


Target 5:


(SEQ ID NO.: 7)


5′-TGATACAAATACTCTCAATCAAAGCCAATATGTCGCAAAA-3′


 


(SEQ ID NO.: 8)


5′-CAAAGTAAAATTAACAGATAGTACGTTCTCAATCTCGCGA-3′


 


Target 6:


(SEQ ID NO.: 9)


5′-TCTTCTTGCACATATTTTTCTCCGTGAGACATGTAAATAA-3′


 


(SEQ ID NO.: 10)


5′-ATGTACTTCACTTCAGTTTTCTTTAAACACGTTTCACATA-3′


 


(SEQ ID NO.: 11)


5′-CCGCATTTATCAGTTTACCGCCCCATAAACATAACCGCT-3′


 


(SEQ ID NO.: 12)


5′-AATTATGCTCATGATTTTCTTCAAAAAGGCTCGCGCAATT-3′


 


(SEQ ID NO.: 13)


5′-GGCTGTTAAACTTACTTTTCTTCAGTAATTGCCGTTGACA-3′


 


(SEQ ID NO.: 14)


5′-TAGTTTATCAGGAGCGATCACTGATCATGAGTAACTTTTA-3′


 


(SEQ ID NO.: 15)


5′-ATCAAGAATTGATAATTTTAGGAATTGCGTATCGCTGCTA-3′


 


Target 7:


(SEQ ID NO.: 16)


5′-AACTGTGTTTTAGGACTTCATTGTCTTAATTCTCTTCCCT-3′


 


(SEQ ID NO.: 17)


5′-AGCGCGATATTACGGTCTCGAACCAAAACCATCACGGTTC-3′


 


(SEQ ID NO.: 18)


5′-ATAGTTTAAATTTAATCTTCTGCCACCCTTCACTTTCA-3′


 


Target 8:


(SEQ ID NO.: 19)


5′-GCACATCTCCCGTTCGACTTTTTTATCTTCGAGCACCTAA-3′


 


(SEQ ID NO.: 20)


5′-CCATGTAGCACAAAACAACGATATAAGAACTACATTTAGT-3′


 


(SEQ ID NO.: 21)


5′-GGCTTTCTAATCTAACACGATCTCCTCTCCTTACGCCGTG-3′


 


(SEQ ID NO.: 22)


5′-TACTCTTGTTTAAACTAGAACAGTAAAATATTAATTCTTA-3′


 


(SEQ ID NO.: 23)


5′-TGGAGTTTATAATACTCGAGGCTAGTAGTGCCATTTTACA-3′


 


(SEQ ID NO.: 24)


5′-ACGCGCGGCTGTGGGGAAGGTACAGGTTCCGAACGATGGA-3′


 


(SEQ ID NO.: 25)


5′-ACAAGAACTATTTTTATCAAAGACGTCACCAACTTAAGGC-3′


 


(SEQ ID NO.: 26)


5′-CAAATAATCGTTTTATAATTACCAACACATTTTGGTTAAC-3′


 


(SEQ ID NO.: 27)


5′-TTTATATTAAGCAACTTTTTGAGAGTTGATTGATAATTTA-3′


 


Target 9:


(SEQ ID NO.: 28)


5′-TGGCCTAATCTCGGAGACTGGCCGCTGTGGGCGCGGGCCT-3′


 


Target 10:


(SEQ ID NO.: 29)


5′-TCTAATTATGTTACAAAATAATTGTTATGCTCCGCAAATA-3′


 


(SEQ ID NO.: 30)


5′-TCATTTAATTGTCCTAAATCTGAAAATTTATTATATTTC-3′


 


Target 11:


(SEQ ID NO.: 31)


5′-CCGGGATAAATTACTAAGTTTCGGGTATTTTGACAATATT-3′


 


(SEQ ID NO.: 32)


5′-AAACCTGCAAAATTTAGGGCCAATGTGTGTATTGAACGGG-3′


 


(SEQ ID NO.: 33)


5′-GTTATAGTAATATTGGTTCTAGCTCTCAGTAATATCAAAA-3′


 


(SEQ ID NO.: 34)


5′-ACCCTATAAGCTGAGATAAGCATTCTGTGGACGAAAAGTT-3′


 


Target 13:


(SEQ ID NO.: 35)


5′-AGGCGTTAACTCTTGTCATGTTATAGACGTCTAATCCATC-3′


 


Target 14:


(SEQ ID NO.: 36)


5′-ACTGTAATCACTTCTTTTAAATAGTCCCGGAACGATATCA-3′


 


(SEQ ID NO.: 37)


5′-CTTGATCCATACTATAACTTAACATTTGTTCATCTCAAGT-3′


 


Target 15:


(SEQ ID NO.: 38)


5′-CTAAATCGCGAACCGAGTTTTTGTCAAAGTTCTAGATTAA-3′


 


Target 16:


(SEQ ID NO.: 39)


5′-ACGAACTTTTTTAATTCGCAGACATGTTTATAGTTTCTTG-3′






No aptamers were recovered for target 12 (IL-7R cat 306-IR).
Example of Aptamers to Target Molecules
Examples of aptamer sequences to target molecules are illustrated in the following table showing, for example, the target molecule and the aptamer sequence in 5′ to 3′ DNA sequence. The sequences may also include any modifications, concatenations, and/or truncations thereto and in general may include any sequence with substantial or significant homology or sequence identity with the aptamer sequence shown in the figures. Unless otherwise notated, the targets in the table are generally human-derived proteins. For example, LAP(TGF-b) stands for latency-associated protein, hCG stands for human chorionic gonadotropin, IgM-BAS and IgM-BAG are both immunoglobulin M, IgC-FC stands for IgG, Fc constant region, and murine monoclonal anti-Hen Egg Lysozyme antibody (an IgG).









TABLE 1






TARGET
5′ to 3′ Sequence







SEQ ID NO.: 40
IgM-BAG
GAGGGGGGGGCCAGGCCGCCAGGAGCGAAGGTCCCGGCCC


 


SEQ ID NO.: 41
IgM-BAG
CGTGGTTGGATTGGGGGGCGTGTTCGCCTGAGTGCAAGGC


 


SEQ ID NO.: 42
IgM-BAG
TACGCGGTTTTTGTATCCCAAACCATTGCATCATCTCTAA


 


SEQ ID NO.: 43
IgM-BAG
GTCATTCTTTTAGTATTAAATTTAGAATTACTCCTCCAGA


 


SEQ ID NO.: 44
IgM-BAG
CAAAGAAAATGGTCATGAAATAGCGTACTAACATGGAGTC


 


SEQ ID NO.: 45
IgM-BAG
CTAGTTTATCTTATAACGAAATGTTGTTTTTATGCTTTCA


 


SEQ ID NO.: 46
IgM-MuChain
TGCAAAAGAGCCCTACTCTTGCTCTCAGATCCCTTCTC


 


SEQ ID NO.: 47
IgM-MuChain
TTCGAATTCTATCAATTTGAGACGATTTAGT


 


SEQ ID NO.: 48
IgM-MuChain
AGGTCGTTTATGACTAACACTTTAGATTCGACACACAG


 


SEQ ID NO.: 49
IgM-MuChain
AGTTGTTTGGTCGATATGGCCTTTGCTCCAGGGTTGCCG


 


SEQ ID NO.: 50
IgM-MuChain
ACAAAAGAAAGGTTGCATCGAACAGATAACTTACATAT


 


SEQ ID NO.: 51
IgM-MuChain
GAGTGAATGTCAGGTGCATGAATGTTTCCGTATAGCGCGA


 


SEQ ID NO.: 52
IgM-MuChain
TAGATTAATTGGATGTTGTATACCTAGTATAGCCATTG


 


SEQ ID NO.: 53
IgM-MuChain
AAGCGCGTTATCAGTATAAAGGAAACATAACATACTCG


 


SEQ ID NO.: 54
IgM-MuChain
TAATTATTTAGTAATAGATTAAGTTTCTTAGATGCTAAC


 


SEQ ID NO.: 55
Collagen
AGACTTGAAAGCATCTTTACTTCGATTGGTAATATTTTTG


 


SEQ ID NO.: 56
IgG-FC
TTGAAGGCGTACCGTCCGCGGGCGGCGTGTGCGCCGGGCC


 


SEQ ID NO.: 57
IgG-FC
TTGAAGGCGTACCGTCCGCGGGCGGCGTGTGCGCCGGGCC


 


SEQ ID NO.: 58
IgG-FC
GGGGGCGCGTCGCAGGGGGGGACGCGGGAATCGAGGAGCA


 


SEQ ID NO.: 59
IgG-FC
GGGGCCGATTCGGCCAGTCCGGGGGGCCCGACATCGGAGA


 


SEQ ID NO.: 60
IgG-FC
CGGAAAATTATTCTGTAATTTTCTAACTCTGGTTAGACTT


 


SEQ ID NO.: 61
IgG-FC
CCAATTTGGGATATGCTTCAGGATCCCCTGAGTATGGTTT


 


SEQ ID NO.: 62
IgG-FC
CAAACGCATTAGATCGAATCTAATTGTTGCAACAAAGTCA


 


SEQ ID NO.: 63
IgG-FC
CAGGGATTTATCCCCCATGCGGACNCGTAGCCACCCGGAA


 


SEQ ID NO.: 64
IgG-FC
CGTTAGTTTTCTTTACGTGAAAACAGTTTGACTTACGCCA


 


SEQ ID NO.: 65
IgG-FC
CGTTTTATTATGGGTTTATAAAACATCAGCATCACAAGAT


 


SEQ ID NO.: 66
IgG-FC
ATTTATAGGGTCTGTATTAAAACAATTTTAATTTCACTCT


 


SEQ ID NO.: 67
IgG-FC
CCACGGGGTGGGATTCTATTATTTAACTAACTAATGTACA


 


SEQ ID NO.: 68
IgG-FC
AAGGGATGTTTGGCGTTCTGATTAACGTTAGGAACCATGT


 


SEQ ID NO.: 69
IgG-FC
TGAAATAAATTCTTGAAGAGAACCATTTATCGGGTCGTCA


 


SEQ ID NO.: 70
IgG-FC
CGGGGGTTCCCTGTAATATAAAGTGTCATTTAGTGCGCCT


 


SEQ ID NO.: 71
IgG-FC
CGAATTTAGTTAATGATCGTAATATTACAAATAAATTT


 


SEQ ID NO.: 72
IgG-FC
GCAATTTTCAGGGTATCAACAGGCCCATATGGATCATCAC


 


SEQ ID NO.: 73
IgG-FC
CGAAGTTGAGTTATTTATTTATCTCATCTAATAGTCAGTT


 


SEQ ID NO.: 74
IgG-FC
CATGGTGTTTATTGATCAATCTTTGACCGTAGAGGAATAT


 


SEQ ID NO.: 75
IgG-FC
GTAGTTTTCAAATTTAAGCGGCGTGAACTTAATAAGTACT


 


SEQ ID NO.: 76
IgG-FC
GGCTTTTATTCGTGCCGTTTAACAGACAAAATCATTCAC


 


SEQ ID NO.: 77
IgG-FC
AGGTATTATTTTACAAAAGAATTAGCTATAACCGAATAGA


 


SEQ ID NO.: 78
IgG-FC
AGGTATTATTTTACAAAAGAATTAGCTATAACCGAATAGA


 


SEQ ID NO.: 79
IgG-FC
TTTAAGTTAATAGTAGTTCTGAAGACGATTACCCCGTGA


 


SEQ ID NO.: 80
IgG-FC
AGCTAATACTTTATTTCTTTAGAGAGTTGTGCATA


 


SEQ ID NO.: 81
IgG-FC
GTGTTACTTTTTTTTCTGGTGAACGAGTTAACTACTTCAA


 


SEQ ID NO.: 82
IgG-FC
GTTGTAGTTTTAAGATTAAGTGTACGCATGTTACGGGTAT


 


SEQ ID NO.: 83
IgG-FC
ATTAATATTTAATCAAGGCTCTCAACATTTTCATACTAT


 


SEQ ID NO.: 84
IgG-FC
AGTACTATTGAGATTATTCGTCATGGAAATCGGTATCGCT


 


SEQ ID NO.: 85
IgG-FC
CGAAAAAACGGTTATTATTATCTTCTTATTATCTCCCTCA


 


SEQ ID NO.: 86
IgG-FC
CGATTTTGGTATTAATTATATATGCGGTGTGGTCGAGGTT


 


SEQ ID NO.: 87
IgG-FC
CGTTTATTTATTGAACCACTTTTGTTATCTAGCGCTTAGC


 


SEQ ID NO.: 88
IgG-FC
CTTATTGTTAAAGGTCTAGTTTTATTTCAATCTCACACCT


 


SEQ ID NO.: 89
IgG-FC
GATAATTTCTAAGGATGCGCTAACATAACTCACTCGTATT


 


SEQ ID NO.: 90
IgG-FC
GTCGTTCGTTCCCTAATCTTTTCTCCTTAGTTCAATTCA


 


SEQ ID NO.: 91
IgG-FC
GTGAGATTTTATAATACTTTAAGCACGTATCCTGATT


 


SEQ ID NO.: 92
IgG-FC
GTTTAATTTCGTTATTTTTCTAAGTTTCAAGATTTGCTCA


 


SEQ ID NO.: 93
IgG-FC
TGTTTGTTGGAATATTTATTTGAAGGTCCGTATATATCTT


 


SEQ ID NO.: 94
Fibrinogen
GGCGCGCTGGCGCGCGAAGGTGGCTCGGAGTGCTCCGGGC


 


SEQ ID NO.: 95
Fibrinogen
GAGGGCGGGCCGGGCCCGGCCCAGGAGGAGGGAGGCCCCG


 


SEQ ID NO.: 96
Fibrinogen
CGGATAGGTAGGCCCCCTCCACGGGCCGGGTCGGCCCGGC


 


SEQ ID NO.: 97
Fibrinogen
CAGGGCGTGAAAGGAGCGGGGCAGGGGCCCCGAAACAAAC


 


SEQ ID NO.: 98
Fibrinogen
GGGTTGTTTTTACGAACCGGACCGAATAACGGCACCGGCC


 


SEQ ID NO.: 99
Fibrinogen
TGGGAGGGCTTGGGAGCCAGGTCGTCAAGGCGGGGTCCCC


 


SEQ ID NO.: 100
Fibrinogen
GCATCACATATCGCCCCGTGACTGGGCCAAAAGGCCGGAC


 


SEQ ID NO.: 101
Fibrinogen
CCAATGAATTGAGTGTGCTTTTTTTTTCGAAACTCAATTC


 


SEQ ID NO.: 102
Fibrinogen
CAGATGGACGTTCGTCGTTAATTGTTAAGGCGTCGCCGTC


 


SEQ ID NO.: 103
Fibrinogen
GATATTCTGAGTGATCGATCGTACGATCAATAATCTAGTA


 


SEQ ID NO.: 104
Fibrinogen
GGGTCGATTGGTTTTGCTTCCATACTATATGTAGCAATTG


 


SEQ ID NO.: 105
Fibrinogen
CGATTTGTAATGGTTTATACCATTTAGGTTTTTCGAAAAT


 


SEQ ID NO.: 106
Fibrinogen
AGCTATTCTATGGAGAGTCAATTTTCACTGCATAGCAGTA


 


SEQ ID NO.: 107
Fibrinogen
CAGTAATATTCTACTTTCCGATACGGCTTCGTATGCGATC


 


SEQ ID NO.: 108
Fibrinogen
AGGATTTAATCTGTGTGATTAGCATGTTCCGACACCGGCC


 


SEQ ID NO.: 109
Fibrinogen
GTGGCAAATTTATGTGTTCTCAAAAAACACAAACAACAAC


 


SEQ ID NO.: 110
Fibrinogen
CGTAACGACCTGAAATTCGGGTACTACAGAAATCCAATTA


 


SEQ ID NO.: 111
Fibrinogen
TTGTTTTATGTTTTTGCCAGACTTTAAGGCACCAAGCT


 


SEQ ID NO.: 112
Fibrinogen
CCGATTATTGTTATTCTAGAAGTGAGCATGATCGCACACT


 


SEQ ID NO.: 113
Fibrinogen
CTTGTTTTTGAAAGCATGTCGCTAAATGGTAGTTTTCAAT


 


SEQ ID NO.: 114
Fibrinogen
CAGTGGAGGGGAGTGCGTTCTGGAGGAGCGGCCCGCAGAC


 


SEQ ID NO.: 115
Fibrinogen
CTAGGATAAAAGGTAATTGAATTAACATAGGCTTTTAACG


 


SEQ ID NO.: 116
Fibrinogen
GGCGGGGGGCTAGCAGAGCGGGAACGGGCGGCGGCAACAA


 


SEQ ID NO.: 117
Fibrinogen
TGTACGGGGGGATCCAAGTTATGGACAGGCTTCAATTAGA


 


SEQ ID NO.: 118
Fibrinogen
CCTTGTCGGTAAATACCAGTACTAGTACGTTAGATACGGA


 


SEQ ID NO.: 119
Fibrinogen
CCTTGTCGGTAAATACCAGTACTAGTACGTTAGATACGGA


 


SEQ ID NO.: 120
Fibrinogen
GAGAAGGGGAGATGGGGAGGGATACACGGGCCCGCATATG


 


SEQ ID NO.: 121
Fibrinogen
AGATTAAAATATGTAGACCCCATGTTAATCAATGAACACT


 


SEQ ID NO.: 122
Fibrinogen
GAGTTTGGAATGTATGTGTATATACACGCCCTTCATTTTT


 


SEQ ID NO.: 123
Fibrinogen
GTCTGAAGACTAGATTTCTTTTTCAAAATGAACAGGCCA


 


SEQ ID NO.: 124
Fibrinogen
ACGAAATATAGTTATTTAGTCCTTATTACATTTTTGCTTC


 


SEQ ID NO.: 125
Fibrinogen
TGTTAATGATTTAAAGACTGTTTGATAACGCAGTAA


 


SEQ ID NO.: 126
Fibrinogen
CAATATAATAAATGTTGAACCGTGTAATTCATAATACGAC


 


SEQ ID NO.: 127
Fibrinogen
GAAGAAATAGCGATGATAATTTCAATTCCGTAAACCAGTC


 


SEQ ID NO.: 128
Fibrinogen
TACATATAAAGATGTGTCAACTAGAAATACTTTCCATACT


 


SEQ ID NO.: 129
Fibrinogen
ATTCTAAGGCTTGAAGCAGTCCTAACCTATAACTCCGGTG


 


SEQ ID NO.: 130
Fibrinogen
AAGGTTTATGAGTAAGTCGGATGCCTACAATATACTTAAT


 


SEQ ID NO.: 131
Fibrinogen
CACTGTTTGCGGAAAGAACTTGATTTGAGTTAGTATACCA


 


SEQ ID NO.: 132
Fibrinogen
TTAAGGTGTAAATTTAAAAATGTTTACCTATTCTTTCCAC


 


SEQ ID NO.: 133
Fibrinogen
CGATTTATTTGGGTAACAGTCCATCCACGTTATACACACG


 


SEQ ID NO.: 134
Fibrinogen
GATAATAGAAAAGCTTACGCACATCTAGAC


 


SEQ ID NO.: 135
Fibrinogen
TAAATATGTCAATTTTAATTCATGCACACCCCTTGACTCG


 


SEQ ID NO.: 136
Fibrinogen
CCGATTATTTATCAATAACATATGAATCCTAACATCCATA


 


SEQ ID NO.: 137
Fibrinogen
GCTATTAGTTGTTGTTCAAATATTCGTACATTCGCTGAAC


 


SEQ ID NO.: 138
Fibrinogen
ATTATCCTTTGTTTTGAATGCATTAGTTACTAACCGCTAA


 


SEQ ID NO.: 139
Fibrinogen
GTTTGTATTCAACAGGCACATGCTATAAGACACTTTACTA


 


SEQ ID NO.: 140
Fibrinogen
CAGGATTTTTTTTGTTCAGGATTATACTTACTTCCTCCCA


 


SEQ ID NO.: 141
Fibrinogen
CGGATTGGCAAAGAGAAGACCAGTTTCTAGGTTATAGTGC


 


SEQ ID NO.: 142
Fibrinogen
CGGGAATGAACGAGGCAGACCACACTAGCGCAGATAGATT


 


SEQ ID NO.: 143
Fibrinogen
GTGATGGATTTTGGACAGCTCAGTTCTAACTCCCAGGAA


 


SEQ ID NO.: 144
Fibrinogen
TAATAATTTATAAATCTGAGGTTTGCATAGTCAACTCTCC


 


SEQ ID NO.: 145
Fibrinogen
CTTGATTAACAGACTATATTTGTTCGAATTACCACACC


 


SEQ ID NO.: 146
Fibrinogen
GTCAGAATTTTTTAGGTTACTTAGGTGACTCCCATATACA


 


SEQ ID NO.: 147
Fibrinogen
AGAGGAAATTTTATTCTGATTTAAGTCATGACCCCCACTG


 


SEQ ID NO.: 148
Fibrinogen
CAGGTANGAGTCGNTAAGTTTTGGTCATCCTNTGCCACT


 


SEQ ID NO.: 149
Fibrinogen
GATGTATTAAGGGGCTTCCACGTTGTGCATGAGTAATTCT


 


SEQ ID NO.: 150
Fibrinogen
TAGAAAAAAAAAGAGTGTACTATTCTACAATAATCTACTT


 


SEQ ID NO.: 151
Fibrinogen
TGGTGTATTTTTTAGCATAACCTTAAGATCTCGGTACATC


 


SEQ ID NO.: 152
Fibrinogen
ACTTTCTTTATCGTCGAATACCTTAATACTGCTCATTGAG


 


SEQ ID NO.: 153
Fibrinogen
CACTGTATACTAACGCATATATTCACATTTGTCATACTTC


 


SEQ ID NO.: 154
Fibrinogen
CATGTAAATTTAAATCTTTGGTAACGGAGTTTTGGCCTTT


 


SEQ ID NO.: 155
Fibrinogen
CGTTCTATCTCATACTTCATCTCCATTG


 


SEQ ID NO.: 156
Fibrinogen
CTAGACAATTGTATTTTTGATGCTTCCACACCCAATTTAC


 


SEQ ID NO.: 157
Fibrinogen
GATGTATTTTCAGCCTAATTCTAAAGTCAATATTTGTG


 


SEQ ID NO.: 158
Fibrinogen
NNNNGTNGTTNGATGANNGNNGATNNNNGGNAGCCTTTAC


 


SEQ ID NO.: 159
IgG-BAS
AGGAGGGGGATCGGGCAGAGGCGGACGGGACGCCCGTGGA


 


SEQ ID NO.: 160
IgG-BAS
GACGGATTTTATAAGGTTATGATATAAACCTCGATCGTTG


 


SEQ ID NO.: 161
IgG-BAS
GTATTGTAAGAGAATCTTTACAACTACAATGTATTTTTAT


 


SEQ ID NO.: 162
IgG-BAS
GGTTTTTTTAAAATCGTTTTTTCATTCAGCAATTAGCTCG


 


SEQ ID NO.: 163
IgG-BAS
GCGTTTTTTCTGATTTTCCTTATTTAATCCACTGATGACC


 


SEQ ID NO.: 164
IgG-BAS
TGTAAGAGATAATTTTAATCGAATTCCTGTGTTATAGCC


 


SEQ ID NO.: 165
Fibrinogen
AGGAAACGGTCTATGTACCAATATTTGTACTATAGGCCC


 


SEQ ID NO.: 166
Fibrinogen
CTAATATTTTAGAAAACTTAGTAAATAGGGCTACTT


 


SEQ ID NO.: 167
Fibrinogen
GTTATTTTATTTAAGCCAAACCTCTAGATACTTCACTATC


 


SEQ ID NO.: 168
IgG-FC
CGTTTAGGTTGCCTAATAAAAATTTCTCCAATTTTACATC


 


SEQ ID NO.: 169
IgG-FC
CTATAAGACATGTTTAAATACAACCTACTGATTGTTATC


 


SEQ ID NO.: 170
IgM-MuChain
GGAGGTTAATTGGGTCAGAGCGTTAACAGGTAACGTTTT


 


SEQ ID NO.: 171
IgM-MuChain
TTTTATTTCGTATCCTATATTTTCAAGTTAGCTTGACTC


 


SEQ ID NO.: 172
IgM-MuChain
TTGATTTTACAAAATGCTTTAAAGTAGGTAATTTGTACCA


 


SEQ ID NO.: 173
IgM-MuChain
CGATTATTGCTTTATAAAAGACCCAGACGTCATCATTATC


 


SEQ ID NO.: 174
IgM-MuChain
TCAATAGTTTTAATCCCTAAACCGACTTCAATC


 


SEQ ID NO.: 175
IgM-MuChain
GGTATAACTCTATTGTCGATAAAATCCCTCTTATTCAGCA


 


SEQ ID NO.: 176
HSA
CTCTTAATGTCACGGCTGAGCCTATGCTGGCGTGACCGA


 


SEQ ID NO.: 177
HSA
GTTTAAATAATGAATACAGGTATGTATTTGGGTCATCCTG


 


SEQ ID NO.: 178
HSA
TGGATCTTTACTTGTTTACTACAAGGTTATTATCGCTTAA


 


SEQ ID NO.: 179
HSA
GTGGGTTTTTTCAAGCTTTTACTGCGCCCGCGTGAGCGT


 


SEQ ID NO.: 180
HSA
TGAATAGTGTCGCGACTGGGGCTGGACCTGCTTGATGG


 


SEQ ID NO.: 181
HSA
TACAAATTGTCTTAATAATCGTTATGTGTATTGGAGTAAT


 


SEQ ID NO.: 182
IgM
GGGGGGGGGCCCGGCCGGCAAGGCCAGTGGCGCCCCGGGC


 


SEQ ID NO.: 183
IgM
GGGGGGGACCGGGGCCGGCCCGGGGCCCCCGCGCCCGGCC


 


SEQ ID NO.: 184
IgM
GAATAAGAAGAATGTCACGCGGCCTTGGGGCCTGCGCCCG


 


SEQ ID NO.: 185
IgM
TAGTGGGAATTAAGTGCGGGTCCGGGCACGGCCTCGCCGC


 


SEQ ID NO.: 186
IgM
AACGGGTATAAGCAGAGATTATGATGAGCCCTCTCTGGCC


 


SEQ ID NO.: 187
IgM
CGCGGAGTGAGTAAAAATTTTAAGCTTATAAACCCGCTTA


 


SEQ ID NO.: 188
IgM
GGTCTTAGAAAACAGATATTCTAGATACTAATATAGTGTT


 


SEQ ID NO.: 189
IgM
CAGTGTATCTTTAGCTGCCGCGGAATTTCCTGAGCCGGAT


 


SEQ ID NO.: 190
IgM
GGTCTAGGTTTATTATCAATATTAGACACAACGGGTATAT


 


SEQ ID NO.: 191
IgM
TCCAAGTTCAAACCTTAGGAACAAATGGATGCGCAGCGAT


 


SEQ ID NO.: 192
IgM
TGATTTTTTATTGATCGTTATTTGAAGACATCTTCCAGGA


 


SEQ ID NO.: 193
IgM
GACGCTTACGCTTGTTAATAAGATTTTTGTTTTCATTACA


 


SEQ ID NO.: 194
IgM
TCTCTTTTCTTGAATCTTGCATTTAACCCATCCCTTCAAA


 


SEQ ID NO.: 195
HSA
GTGTGAAAGCTGGGAGAGTCTGCGGGCCTGTGTCGCGCAA


 


SEQ ID NO.: 196
HSA
CGTAGGAGGGAGATTCCCACAAACGCTCCCCAT


 


SEQ ID NO.: 197
HSA
CGAGATCGTTATATAAGGGACAATCTGACGATTCTACCTT


 


SEQ ID NO.: 198
HSA
ATGGGTGTGTCTGGGTAGACGTTGTTTTGGCCTGGTGTTA


 


SEQ ID NO.: 199
HSA
TCAATTAAGATCTTGTGTCAAGTGTTAAAGTCCGTCATGA


 


SEQ ID NO.: 200
HSA
AATTGTTCGGTTGACGCTTTTCTGACGCTGTATACCCTGG


 


SEQ ID NO.: 201
HSA
GATGCTATTTTTGATAGATACATGTAACCTTTTAGACTTT


 


SEQ ID NO.: 202
HSA
GATGCTATTTTTGATAGATACATGTAACCTTTTAGACTTT


 


SEQ ID NO.: 203
HSA
AGCTTTTTATGGAATTATTCTCACAACACATTGGAACATT


 


SEQ ID NO.: 204
HSA
TGGAGTGAGTGACTTGACTACTTACAGTAACCTCTACAGT


 


SEQ ID NO.: 205
HSA
ATGTGTCAAAGATTTATCGAGAAACGCTGTTTTTATTGTA


 


SEQ ID NO.: 206
HSA
AGATCCATTAGAATCAATTTATTTGGGCATCGTATTCCGC


 


SEQ ID NO.: 207
HSA
ATTAACTTAAAAACAATCCTTAATCGTTGCAATTAAATCC


 


SEQ ID NO.: 208
HSA
CTTCGTTAAATCTGTATGTACCCGTAGCTAGCTTAATTTC


 


SEQ ID NO.: 209
HSA
CTTTTATCTTCTTATATTGTCCAAGGTCGTATGCAAGCG


 


SEQ ID NO.: 210
HSA
GATTAATCAGTATTCCCGTTCGTTTCTGGCAACATTTACA


 


SEQ ID NO.: 211
HSA
TAGGAATCGGATTATGAAATTGTGGCCCAGGTATCGTCA


 


SEQ ID NO.: 212
HSA
TGATTTTTTGAGGGTTAACTAATTTATATCTGTGTTTT


 


SEQ ID NO.: 213
Hemoglobin
AGGGTCGTTGGGGCCAGGGTTCACGCGCCGCTCCCCGCT


 


SEQ ID NO.: 214
Hemoglobin
GCTGATTCGTTCAGATCTCTATTCTCCTTATTATCGACA


 


SEQ ID NO.: 215
Hemoglobin
GTGATAGGGAAGTGAGTGCTGGCCCGTAGCGACCCTGGAA


 


SEQ ID NO.: 216
Hemoglobin
CAGGAGCGTAATAATCTCGAGAACGTGTGGCAAACGATAC


 


SEQ ID NO.: 217
Hemoglobin
AATGGTGATGATTCTCGTTATTCGTTCAGCCTCTA


 


SEQ ID NO.: 218
IgM-BAS
CGGCTGTCCCGGCCAGGGGGCGGGGCGCGGTGCGCTAAT


 


SEQ ID NO.: 219
IgM-BAS
GCGGGCGCGCAGCGCCACGGGACCGGCCCGCCGGGGGGC


 


SEQ ID NO.: 220
IgM-BAS
CCAATGTGGTGAATAGGAATGTTTCACCGCTTAGGATAAA


 


SEQ ID NO.: 221
IgM-BAS
CGGGGAAATTAGGGAGGGTATCTTCGTTGGTCCTCCGGCC


 


SEQ ID NO.: 222
IgM-BAS
GTTCGTCAAAAATAGAGTGTTTTATGACACAGGAATCCGA


 


SEQ ID NO.: 223
IgM-BAS
GAAAATGGTATATTCGAGTTCTGTGGCATATGGGGCCAT


 


SEQ ID NO.: 224
IgM-BAS
GTTTGTTTCTTTACGGCATGGGTCATCTATCCCAATTACC


 


SEQ ID NO.: 225
IgM-BAS
CTACAAAATTGACAAATCTACTTTTGTGTATTCAAGTTAT


 


SEQ ID NO.: 226
IgM-BAS
GAAGGACTGAAAGAGGACGGAGCGTAGCGGCGTACAGAAC


 


SEQ ID NO.: 227
IgM-BAS
ATGTGTAATTTTTTTACCAAAGCCAAAGCATTTTCCAATG


 


SEQ ID NO.: 228
IgM-BAS
AGGATTGTAATATTGATATCCTGATTCGTTTAATTTGAGC


 


SEQ ID NO.: 229
IgM-BAS
TCCCGTATAGTTACTATTCTTTTATTACTGAATAAGCGAA


 


SEQ ID NO.: 230
IgM-BAS
CTGTTCTATCTTTTCAAGAATGTCCCATCAGTCAATGCCG


 


SEQ ID NO.: 231
IgM-BAS
TCGTCTGTGTCTCAAAAGTGTATCTAGTGATGCCCCAGAT


 


SEQ ID NO.: 232
IgM-BAS
GATAATTTATGGTTAACGAGTTCTTCAGTTGAGGGATTT


 


SEQ ID NO.: 233
IgM-BAS
TGTTCTATTTAATGATTTGTCAACACGATCGGATCTACTG


 


SEQ ID NO.: 234
IgM-BAS
TTCTGATTGGGTCTTTTGATGTTTTATGAAATCATGCA


 


SEQ ID NO.: 235
IgM-BAS
GAGTTTTTTAAAAGAACAGTTTCATCTCCTCAGTCTTAC


 


SEQ ID NO.: 236
IgM-BAS
GTTCATATTTAATCTACTGTATTCCTATTATATGTTTAGG


 


SEQ ID NO.: 237
IgM-BAS
ATCTGGATATAATTAAGTGGGTCAACAGA


 


SEQ ID NO.: 238
IgM-BAS
ATTTTCAAGTATTAACATTATTAGATAGTTTCAAGAGCC


 


SEQ ID NO.: 239
IgM-BAS
GATATTTTGAAATGATTATCCTAGACATCTGATTAGCTAT


 


SEQ ID NO.: 240
IgM-BAS
GGAAAGGAGAAAAGAGGGGAGCAGTGAGTCGTATTA


 


SEQ ID NO.: 241
HyHel5_IgG
ATATAGCGCAACCGAGGGGTAGGACGTGCACCCCAGAGCC


 


SEQ ID NO.: 242
HyHel5_I9G
GGGGGATATTCAAGTCTCCCCTCATTGTATCCCTACCCTT


 


SEQ ID NO.: 243
HyHel5_I9G
ATAAACATGAAGGGGTGGCGCTGGGCAGTCATAATTGAAC


 


SEQ ID NO.: 244
HyHel5_I9G
TTCCAGCGAGGTGGTGCTTAATGAGTCCCGAAAATGTTCT


 


SEQ ID NO.: 245
HyHel5_I9G
GTCGGGAGTAGAGTGGAAGCGAGGAAGGGGGCAAAACACA


 


SEQ ID NO.: 246
LAP(TGF-b)
CCGATGTAGGAGTGAGGCCTAGGTTGACTGCGAGACGCTAACCC


 


SEQ ID NO.: 247
LAP(TGF-b)
CATGACAACTAGGTTTCAAAAGGTCTTTAGATAAAGTCCC


 


SEQ ID NO.: 248
LAP(TGF-b)
CCGCGGGCTTCCAGTATCTCTGGGTACACTACTGGTCAGT


 


SEQ ID NO.: 249
LAP(TGF-b)
GCGAGGATGTCCAAATGCATGGAAAGTAACAGCTCCAAGC


 


SEQ ID NO.: 250
LAP(TGF-b)
CCGATGTAGGAGTGAGAACGACAAACAATCCTTGAGCTGCAATC


 


SEQ ID NO.: 251
LAP(TGF-b)
CCGATGTAGGAGTGAGGACACATGTGAAAAGACATAATTTATTGGG


 


SEQ ID NO.: 252
LAP(TGF-b)
CCGATGTAGGAGTGAGACAACCTGTCATTGACTTCTTAGCTA


 


SEQ ID NO.: 253
LAP(TGF-b)
ACCATCTCTATTGTTGGCACAAATTTGGCCTGCTACATTC


 


SEQ ID NO.: 254
LAP(TGF-b)
CCGATGTAGGAGTGAGAAGACGGGCATTGGATATACCAGCTTATTCAA


 


SEQ ID NO.: 255
LAP(TGF-b)
CCGATGTAGGAGTGAGAAAAACAATCGACCCTATATACCAGCTTATTCAA


 


SEQ ID NO.: 256
LAP(TGF-b)
CCGATGTAGGAGTGAGATGCGTGTTATACCAGCTTATTCAA


 


SEQ ID NO.: 257
LAP(TGF-b)
GCCGTCTTCGATGTGTATCTGCTATGTTAAGGGGACGAGG


 


SEQ ID NO.: 258
LAP(TGF-b)
CCGATGTAGGAGTGAGACTAACCCCGTATACCAGCTTATTCAA


 


SEQ ID NO.: 259
LAP(TGF-b)
GTAAGTCAAACAGTCATCTATCATTCTTATGTCCACTTTT


 


SEQ ID NO.: 260
LAP(TGF-b)
GCCTCTTTGACGTGATGTTCGCTCTTATGACCACATTCAT


 


SEQ ID NO.: 261
hCG
TTTTCATTGCTACAAAGTCATTTTGTAGGTAACGGTGGAT


 


SEQ ID NO.: 262
hCG
ATCTCGGGTGGCCCTTCTAGTGGGAGCATCTCCACTGAAA


 


SEQ ID NO.: 263
hCG
TTTCGCGTATATCACGTCGTATTCAGGAGTAACATTCTAA


 


SEQ ID NO.: 264
hCG
CACAATCAATGTAACATTGCCAATAGTAAATTGAAATCCT


 


SEQ ID NO.: 265
hCG
CCGATGTAGGAGTGAGGTGCATTCCCGGCTCGTATACCAGCTTATTCA


 


SEQ ID NO.: 266
hCG
CCGATGTAGGAGTGAGCGCCGAAAACTGCGAAAGCGACACCG


 


SEQ ID NO.: 267
hCG
CCAATGTAGGAGTGAGAGAAAGCGCGGCTGATATACCAGCTTATTCAA


 


SEQ ID NO.: 268
hCG
CCGATGTAGGAGTGAGGGACGCCTATACCAGCTTATTCA


 


SEQ ID NO.: 269
hCG
CACCCAATGGGGTAAGAGTTGGAATTTACTAACCACCGGA


 


SEQ ID NO.: 270
hCG
CCGATGTAGGAGTGAGGGTGCAATGGTACTGCCCTTCCCTTGG


 


SEQ ID NO.: 271
hCG
AACGGAAAAGTCATACGCGCTTACGATATCGGTTGTCGTA


 


SEQ ID NO.: 272
hCG
TCCGCGAATCTTATAACGGTTCTTCCCTAATGTACATAGG


 


SEQ ID NO.: 273
hCG
CTCATTTAATATAAATNGGATTAGGTGAAAAGTTTCGCTA


 


SEQ ID NO.: 274
hCG
ATCCACCAAAACGGAGTTGCTCGTAATTTATTCATCAACT


 


SEQ ID NO.: 275
hCG
CCGATGTAGGAGTGATACGCTGTGTGTGGCACCAACA


 


SEQ ID NO.: 276
hCG
ACTGAGGTCTGTCCGTTTACTATGTGAAGGTCCAATAATC


 


SEQ ID NO.: 277
hCG
CCGATGTAGGAGTGAGGCTAACCCCGTATACCAGCTTATTCAA


 


SEQ ID NO.: 278
hCG
TGTGTAGAGAATCCCGAGTTTGCACGATGTTCCCTAGCGC


 


SEQ ID NO.: 279
hCG
CCGATGTAGGAGTGAGGGGACATATAACCTATACCTATACCAGCTTATTCAA


 


SEQ ID NO.: 280
hCG
CCGATGTAGGAGTGAGGGTTGAATTGGTTATCGAGACATTGGCG


 


SEQ ID NO.: 281
hCG
CCACAGTTCCAATGTTCTTTATACTCGCGTTGAATCTAAG


 


SEQ ID NO.: 282
Hemoglobin
CGTAGGACACCCTCAAGAAAAAGGGTATTGACCCGGGATAT



(glycated)



 


SEQ ID NO.: 283
Hemoglobin
CCCGTAATTCGCTAATTGCTAGATAACTAGAATCGACTCA



(glycated)



 


SEQ ID NO.: 284
Hemoglobin
GTGAACGGATATCTTTATTCGGCATCTTAGGTAGTCTTAA



(glycated)



 


SEQ ID NO.: 285
Hemoglobin
TTCATTCATTAGCAGACCCAACTGTAATTCAGCCTGTATG



(glycated)



 


SEQ ID NO.: 286
Hemoglobin
CCGATGTAGGAGTAAGACCGCGTGTATACCAGCTTATTCAA



(glycated)



 


SEQ ID NO.: 287
Hemoglobin
TTTGCTATGACATAAAAGGATTTTCGAACAGGAGGCCCAA



(glycated)



 


SEQ ID NO.: 288
Hemoglobin
CCACTTGTAATTTCGATACATTGCGTACTTTCTGCAGGCA



(glycated)



 


SEQ ID NO.: 289
Hemoglobin
CTGAAGTGGCCTTAACCTCAGTGGCAATTTGTAAAAGTA



(glycated)



 


SEQ ID NO.: 290
Hemoglobin
TTGCTCGCTAAATTTGTTTATGCCTCTTTTTGCCAGTATA



(glycated)



 


SEQ ID NO.: 291
Hemoglobin
CTCGATCCGGATAAAAAGCATCTTCCACTCTTTCTACTAA



(glycated)



 


SEQ ID NO.: 292
Synuclein,gamma
GATTATAATTATTAATTATTGTCACGGTAAGTCCAAAGTC


 


SEQ ID NO.: 293
Synuclein,gamma
TCGCATTTAGATAATTGTCATTTTACGACTTCATACCTTA


 


SEQ ID NO.: 294
Synuclein,gamma
GGATGTTTAACGGTTGTCTATATCCCTCTTACACCAATCA


 


SEQ ID NO.: 295
HER2
CTTGATTTTTAATGACTCAGTAAAATGTC


 


SEQ ID NO.: 296
Peroxiredoxin4
CGGTTTATGGTCGTAAAAACTTTACGCTTACCCTTCTTTT


 


SEQ ID NO.: 297
Peroxiredoxin4
GTGTTTTGAATTTATTAAATTGGAAACTACCCGTGCACTT


 


SEQ ID NO.: 298
Peroxiredoxin4
CGTAAGAGGGAGATTCCTACAAACGCTCCCCATCC


 


SEQ ID NO.: 299
Peroxiredoxin4
GGTCTTTTTTTTTTTGAATACTTGGGTCGAGTTTCGCCA


 


SEQ ID NO.: 300
Peroxiredoxin4
CGATTTTTATTGTAATCCATTGGTCACCAACGGTTCAAGA


 


SEQ ID NO.: 301
Peroxiredoxin4
AAGGTTTTTAACCCTCTCGAAAAAGTATCATCCTCAATCC


 


SEQ ID NO.: 302
Peroxiredoxin4
GCGTTAAATGAATAATTCTTTTTAATTTCTTTTACTTG


 


SEQ ID NO.: 303
Peroxiredoxin4
GCGGAATGATTTGTTTTAATACGTCGACAGCATTGCAA


 


SEQ ID NO.: 304
Peroxiredoxin4
GAATTTTTTTCTTAAAAGCTAATTTCCCTTCGCTCACATC


 


SEQ ID NO.: 305
Peroxiredoxin4
CGATTTTTTGGAATAAGTCACTGTGAATGGAAACATAT


 


SEQ ID NO.: 306
Peroxiredoxin4
TGTTAAGATAATTAAGTGTCACCGTCTATACTAAATTT


 


SEQ ID NO.: 307
Peroxiredoxin4
TAGTTGTTTATTTATTCTCATGTTTCGGAGCGTTAACT


 


SEQ ID NO.: 308
Peroxiredoxin4
CAAAGATTTGATAGTTAACGGTTATTGATTTTCACTCTC


 


SEQ ID NO.: 309
Peroxiredoxin4
AATTTTTCGAGTTATGAATATTTCGCCTCTTACTCTTT


 


SEQ ID NO.: 310
Interleukin18
GTATTTTTTTGGTTGTAAAAAAAAGTATCACACTAATTTG


 


SEQ ID NO.: 311
Interleukin18
GGAAAGGGGAAAAGGGGGGAGCGGTG


 


SEQ ID NO.: 312
Crystalin,alphaB
CGTAGGAGGGAGTTCCAATGATACATCCTAACCGATAC


 


SEQ ID NO.: 313
Crystalin,alphaB
GTTCTTTTTTTTACACTAACGGTTTAGTAAACTCTTCGCC


 


SEQ ID NO.: 314
Crystalin,alphaB
GTTCTTTTTTTTACACTAACGGTTTAGTAAACTCTTCGCC


 


SEQ ID NO.: 315
Crystalin,alphaB
AATAATTATGTTCAGCGATACTTCTATTTCCAACTAGCG


 


SEQ ID NO.: 316
Fascinhomologl
CAGTTTTATGTTGGTTTAATCCTGGGGCATAGCGCGTTTT


 


SEQ ID NO.: 317
Fascinhomologl
GTTATTTCTTAAAATATAATACTTC


 


SEQ ID NO.: 318
Fascinhomologl
CGCTTAAAATTTCTCTGTTTTCTGGTAGTAGCGCAATAAG


 


SEQ ID NO.: 319
Fascinhomologl
GTTCTTTATTAAGATGTATTCTATAAGTATTTCAAGTTAA


 


SEQ ID NO.: 320
Fascinhomologl
CAAAAGATTTTAGTAACATCTAGATGGCACGTGATATTTC


 


SEQ ID NO.: 321
Fascinhomologl
TCCTTTTCAATATTTCTTCAACTGAACCTTCGTCATTCA


 


SEQ ID NO.: 322
Fascinhomologl
GGAATATTTTATGGCACTTATTAAACAATTGGTCAAAGTC


 


SEQ ID NO.: 323
Fascinhomologl
GGTCTTCTTTGAGTATTCCTAGTTCTTTGGGGCATTAGTA


 


SEQ ID NO.: 324
Fascinhomologl
NGAGTTTNNGTTTTTAGACATTTTTACCTAACTAGCACGTA


 


SEQ ID NO.: 325
Chloride intercellular
CCATGTTATTTTAATCCTATTTTCAGTACGACTATTACCT



channel1



 


SEQ ID NO.: 326
Glutathione S
GTTAGTAACGGTCAGTTTAATTAAGAACATTTGCTACGAC



transferase pi



 


SEQ ID NO.: 327
Ribonucleotidereductase
CTAATGATGGTTTTCGCAATTAACGCCATCGAACAAGATC



M2polypeptide



 


SEQ ID NO.: 328
Ribonucleotidereductase
CTTATTTAATTGACTTTTAGTAAATGTTTTTCAGTTTTAA



M2polypeptide



 


SEQ ID NO.: 329
Ribonucleotidereductase
CTAATTTTAAATCAGTATTTTTTTCATTCTATCGCACTAT



M2polypeptide



 


SEQ ID NO.: 330
Ribonucleotidereductase
GTCTGATCTCTTTGAATCTTTTACCGCATATACTGTTCGT



M2polypeptide



 


SEQ ID NO.: 331
Clusterin
ACCATTTGATGGTTTTCCCTAATTACCAGTTTAATATTAA


 


SEQ ID NO.: 332
Clusterin
CGGATTTTTAGAGTCTTGAAATAGTTTTCTGTCTCCAGAC


 


SEQ ID NO.: 333
p10
CGTAGGAGGGAGATCCCTACAAAC


 


SEQ ID NO.: 334
pll
CTTTTTTATGAATTCCCTTTAACGCTCTTTGATACATTC


 


SEQ ID NO.: 335
p12
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 336
p13
CGTAGGAGGGAGGTTCCTGCAAAC


 


SEQ ID NO.: 337
p14
CGTAGGAGGGAGATTCCTACGAAC


 


SEQ ID NO.: 338
p15
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 339
p16
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 340
p17
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 341
p18
CGTAGGGGGGAGATTCCTACAAAC


 


SEQ ID NO.: 342
p19
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 343
p2
CGTAGGAGGGAGATTCCTGCAAAC


 


SEQ ID NO.: 344
p20
CTATTCTTGGTTTAACGGCTTATTATAACC


 


SEQ ID NO.: 345
p21
CGTAAGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 346
p22
CAAGGGTTTTTAAGTGGTTCGGCGAAGTGACACGTCGTTT


 


SEQ ID NO.: 347
p23
CGTAGGAGGGAGATTCCTACAAGC


 


SEQ ID NO.: 348
p24
GTTTAAAATTATTAACTGTGTTGTCCTAGTCTTGTTCA


 


SEQ ID NO.: 349
p25
GTTTAAGTGGTTATTGAGACATTTTTAATCCGAAATC


 


SEQ ID NO.: 350
p26
GTGATTTATTAGGAATCAAGTCTAAGAGCATAT


 


SEQ ID NO.: 351
p27
CGTAGGAGGGAGATTCCTACAAGC


 


SEQ ID NO.: 352
p28
CTTTTTTAAGTTGAGTATATGGGTAAA


 


SEQ ID NO.: 353
p29
GGATATCTTTTTTTGATACTCTGATGAATC


 


SEQ ID NO.: 354
P3
CGTAGGAGGGGGATTCCTACAAAC


 


SEQ ID NO.: 355
p30
CGTAGGAGGGAGATTCCTACAAACGCTCCCCA


 


SEQ ID NO.: 356
p31
CGAAATAGTTTTAATTGTTGTATCCCG


 


SEQ ID NO.: 357
p32
NNTGATTTATTAGGAATCAAGTCTAANAGCATAT


 


SEQ ID NO.: 358
p33
CGAAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 359
p34
TCGATTTTGTATAATTCTTTATACCCTTTGGTCTTGTC


 


SEQ ID NO.: 360
p35
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 361
p36
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 362
p37
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 363
p38
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 364
p39
CGTAGGAGGGAGATTCCTGCAAAC


 


SEQ ID NO.: 365
p4
CGTAGGAGGGAGGTTCCTACAAAC


 


SEQ ID NO.: 366
p40
CGTAGGAGGGAGATTCCTGCAAAC


 


SEQ ID NO.: 367
p41
GGGAGGGAGGGGGCGACGGCCAGGAGCG


 


SEQ ID NO.: 368
p42
CGTAGGAGGGAGATTCCTACAAACGCTCTCCATCC


 


SEQ ID NO.: 369
p43
CGTAGGGGGGAGATTTCTGCAAAC


 


SEQ ID NO.: 370
p44
CGTAGGAGGGAGATTCCTGCAAAC


 


SEQ ID NO.: 371
p45
CGTAGGAGGGGGATTCCTACAAAC


 


SEQ ID NO.: 372
p46
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 373
P5
CGTAGGAGGGAGATTCCTACAAGCACTC


 


SEQ ID NO.: 374
p6
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 375
P7
TCTAATATGTTTTATAAACTCGGTTTTACCGTCTCG


 


SEQ ID NO.: 376
p8
CGTAGGAGGGAGATTCCTACAAAC


 


SEQ ID NO.: 377
P9
TGATCTTATTTAGAAACTCCCTTCCGTTGGGAGGGACCAG


 


SEQ ID NO.: 378
HyHel5IgG
CCGATGTAGGAGTGAGGAGAGC


 


SEQ ID NO.: 379
LAP(TGF-b)
CCGATGTAGGAGTGAGGTCTTGCCTCGGGATTACAGATGCGCCCG


 


SEQ ID NO.: 380
LAP(TGF-b)
CCGATGTAGGAGTGAGTAATGATCAAAGTCAGGAACCGCGTTCCC


 


SEQ ID NO.: 381
LAP(TGF-b)
CCGATGTAGGAGTGAGCCGGAT


 


SEQ ID NO.: 382
hCG
CCGATGTAGGAGTGAGGGCTCCAGTATTCTATACCAGCTTATTCAA


 


SEQ ID NO.: 383
hCG
CCGATGTAGGAGTGAGGTGGACTAACCATATACCAGCTTATTCAA


 


SEQ ID NO.: 384
hCG
CCGATGTAGGAGTGAGGTGGACTAACCATATACCAGCTTATTCAA


 


SEQ ID NO.: 385
hCG
CCGATGTAGGAGTGAGGAAGATTTCCACTATACCAGCTTATTCAA


 


SEQ ID NO.: 386
hCG
CCGATGTAGGAGTGAGGATACGTTCGAATGGCTTACATCATACCCC


 


SEQ ID NO.: 387
hCG
CCGATGTAGGAGTGAGGCTAACCCCGTATACCAGCTTATTCAA


 


SEQ ID NO.: 388
hCG
CCGATGTAGGAGTGAGGCTAACCCCGTATACCAGCTTATTC


 


SEQ ID NO.: 389
hCG
CCGATGTAGGAGTGAGGCTAACCCCGTATACCAGCTTATTC


 


SEQ ID NO.: 390
hCG
CCGATGTAGGAGTGAGACCGCTG


 


SEQ ID NO.: 391
hCG
CCGATGTAGGAGTGAGGGCCTGTTTTATACCAGCTTATTCAA


 


SEQ ID NO.: 392
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 393
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 394
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 395
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 396
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 397
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 398
hCG
CCGATGTAGGAGTGAGCCCAGT


 


SEQ ID NO.: 399
Hemoglobin(glycated)
CCGATGTAGGAGTGAGGCTGGCAGTATACCAGCTTATTCAA


 


SEQ ID NO.: 400
Hemoglobin(glycated)
CCGATGTANGAGTGAGCTAATAAA


 


SEQ ID NO.: 401
Fibrinogen
CAATATGTCTAATTTTTTTACATGGCGGCATGGTATTGGC


 


SEQ ID NO.: 402
Fibrinogen
CAATACAATACTATATTTGTGTCAATCTCGTACTTCTGAC


 


SEQ ID NO.: 403
Fibrinogen
TAATTATCTCCTTAATCATGGTTATTCTTTGAATCTATCA


 


SEQ ID NO.: 404
Fibrinogen
ATAGTCTAATACAACTTAAAGCAATTCCATGATTATAAAT


 


SEQ ID NO.: 405
Fibrinogen
ATTCGTTTACATTATTCGGCAATTCTTATTTCTGTTGGAG


 


SEQ ID NO.: 406
Collagen
AGAGGCGGCTGAGATCAATCTCCGCTCAGGGAGCGAGTA


 


SEQ ID NO.: 407
Collagen
GTAATAGGTGATTTCCTCAATTTGAATTAGATCACAAAAT


 


SEQ ID NO.: 408
Fibronectin
CATGTGATGCTCACGGTGGCACCCCAGGCGAGTACGCAGT


 


SEQ ID NO.: 409
Fibronectin
GATGGTGTTTGTACACAACTTTACATTTTAGTCCTACAAG


 


SEQ ID NO.: 410
Fibronectin
CAAGAGTGTTAGACATTATCTCAGCGCTGCCAATTATATT


 


SEQ ID NO.: 411
Fibronectin
CCTTGCGACAAAACCCTCGGGACCTCTATCAAGCCAACGT


 


SEQ ID NO.: 412
Acetyl-BSA
ACCATATGAATACAACACCATTCAGTTTATTATCCTTTT


 


SEQ ID NO.: 413
Acetyl-BSA
AAGATCTTTATTAAGCAAACAATGTTAACTATAGAGCGTT


 


SEQ ID NO.: 414
Acetyl-BSA
GAATTACATTCAAAAATTTTCTTCTGGCATCTGTAATACC


 


SEQ ID NO.: 415
Acetyl-BSA
ACAATGTATAATTATATCGATTCAGATTAGTCTACAGGAC


 


SEQ ID NO.: 416
Heparan
CACAGAATGTGGATATTTTCTTGCATCTCTTCCTTTTAGT



Sulfate



 


SEQ ID NO.: 417
Heparan
TAGCGCAATTCGTAGTTTCAGGTATCTGGATTCAGGCCGT



Sulfate



 


SEQ ID NO.: 418
Heparan
GTAATCGCGTTACTACTATCTCTCCGTCCACTTTCAATAC



Sulfate



 


SEQ ID NO.: 419
Heparan
CAAAGTAAAATTAACAGATAGTACGTTCTCAATCTCGCGA



Sulfate



 


SEQ ID NO.: 420
Heparan
CTGGGCATTTCTAAGGAGTCATACAACTATTTCAGGTTAT



Sulfate



 


SEQ ID NO.: 421
Heparan
TGATACAAATACTCTCAATCAAAGCCAATATGTCGCAAAA



Sulfate



 


SEQ ID NO.: 422
PIF-HALSA-
GCAGGGCGCGACTCGGCGTGGAACGAGGTTCAATAGTCCA


 


SEQ ID NO.: 423
PIF-HALSA-
ATCAAGAATTGATAATTTTAGGAATTGCGTATCGCTGCTA


 


SEQ ID NO.: 424
PIF-HALSA-
TAGTTTATCAGGAGCGATCACTGATCATGAGTAACTTTTA


 


SEQ ID NO.: 425
PIF-HALSA-
GTTTAGTTAAAATCCGTTTGAGAACAAATTACAAACCTTA


 


SEQ ID NO.: 426
PIF-HALSA-
AAAAGTCGTAATAGCCCGGGACAACGCCAGCTAAAAGAAA


 


SEQ ID NO.: 427
PIF-HALSA-
CCGCATTTATCAGTTTACCGCCCCATAAACATAACCGCT


 


SEQ ID NO.: 428
PIF-HALSA-
ATGTACTTCACTTCAGTTTTCTTTAAACACGTTTCACATA


 


SEQ ID NO.: 429
PIF-HALSA-
CGTCAGTCTGCTTTCTTGGCTTGTGTACTTAATAATAAGG


 


SEQ ID NO.: 430
PIF-HALSA-
AACCAGTAAGGTCAGAGTAATAGTATGCCAGTCTTGATCT


 


SEQ ID NO.: 431
PIF-HALSA-
AATTATGCTCATGATTTTCTTCAAAAAGGCTCGCGCAATT


 


SEQ ID NO.: 432
PIF-HALSA-
AGAATTTTTAAGGGTTATCTCAAGTCTTGAACATCTAACG


 


SEQ ID NO.: 433
PIF-HALSA-
GGCTGTTAAACTTACTTTTCTTCAGTAATTGCCGTTGACA


 


SEQ ID NO.: 434
PIF-HALSA-
TCTTCTTGCACATATTTTTCTCCGTGAGACATGTAAATA


 


SEQ ID NO.: 435
PIF-HALSA-
CGTCTAATCAATATTGTTTAATGTATTTTGCCAGACACTA


 


SEQ ID NO.: 436
PIF-HALSA-
CCCAGAATGTAGCTTACCTTTTTTGATCGTCCCAGTCCTT


 


SEQ ID NO.: 437
RnaseA
GTGTGCCCGGTCAACGCGTGGGCCGCGTGGTACGGGGCGT


 


SEQ ID NO.: 438
RnaseA
AGCGCGATATTACGGTCTCGAACCAAAACCATCACGGTTC


 


SEQ ID NO.: 439
RnaseA
ATCGACTTAATTTAAAGTGAAAAGATCCCTTTCCACAAAT


 


SEQ ID NO.: 440
RnaseA
TCGTTATTAGGTTGAGTAACCCATTCTCTTAGCCGCTATA


 


SEQ ID NO.: 441
RnaseA
TGGTGTTTTACAAAATGAGTACGTTTTTAATCTCGCCCGG


 


SEQ ID NO.: 442
RnaseA
CCAGGGTACACATCACGAAATATCTAACCTGATTGCAAAC


 


SEQ ID NO.: 443
RnaseA
TATCGTTTAGTTTACAACTTTCAAATTTAATAAATCGAAT


 


SEQ ID NO.: 444
RnaseA
AACTGTGTTTTAGGACTTCATTGTCTTAATTCTCTTCCCT


 


SEQ ID NO.: 445
RnaseA
CGTATATATAGGACGTTTTTGACAGTTTTATTTATTAAAT


 


SEQ ID NO.: 446
RnaseA
CGTTCATTGTTGGTATAGTTAAGTTCTGACAGATCAATAA


 


SEQ ID NO.: 447
RnaseA
ATAGTTTAAATTTAATCTTCTGCCACCCTTCACTTTCA


 


SEQ ID NO.: 448
Laminin
CCCTGGCCAGGCGGGCGCCCGGCCGCGGGCGTGGGGGACG


 


SEQ ID NO.: 449
Laminin
TTTATATTAAGCAACTTTTTGAGAGTTGATTGATAATTTA


 


SEQ ID NO.: 450
Laminin
GGTGGATAACTGTGTCTGCTTGCCAGACTACGTCCTCAGA


 


SEQ ID NO.: 451
Laminin
ACGCGCGGCTGTGGGGAAGGTACAGGTTCCGAACGATGGA


 


SEQ ID NO.: 452
Laminin
AAAAGAGAGGAACCGGTCTTGGCCTGCTCTAAGATTTTGT


 


SEQ ID NO.: 453
Laminin
CCGATATTGGATCTAAGTGTTGCATCAACATTAATTCAGA


 


SEQ ID NO.: 454
Laminin
TTCCTTCGTCTTAATACTGTTGCCAGTTAATTAATTTGCG


 


SEQ ID NO.: 455
Laminin
ACAAAGGATGATCTTCTTATCCTTCAACTAGATCCGGTCC


 


SEQ ID NO.: 456
Laminin
TGGAGTTTATAATACTCGAGGCTAGTAGTGCCATTTTACA


 


SEQ ID NO.: 457
Laminin
CAAATAATCGTTTTATAATTACCAACACATTTTGGTTAAC


 


SEQ ID NO.: 458
Laminin
ACAGCTCTCACGCTCCGTCAAGACCAATTTCCATTCGGTT


 


SEQ ID NO.: 459
Laminin
GCACATCTCCCGTTCGACTTTTTTATCTTCGAGCACCTAA


 


SEQ ID NO.: 460
Laminin
CCATGTAGCACAAAACAACGATATAAGAACTACATTTAGT


 


SEQ ID NO.: 461
Laminin
TACTCTTGTTTAAACTAGAACAGTAAAATATTAATTCTTA


 


SEQ ID NO.: 462
Laminin
GGCTTTCTAATCTAACACGATCTCCTCTCCTTACGCCGTG


 


SEQ ID NO.: 463
Laminin
TAAAATGGATGTTTTGAAAATTCTGGTATCTCGAGTGTC


 


SEQ ID NO.: 464
Laminin
TATACATTGAGATAAAACCGATCTTGAAATTTTCCGCACG


 


SEQ ID NO.: 465
Laminin
ACAAGAACTATTTTTATCAAAGACGTCACCAACTTAAGGC


 


SEQ ID NO.: 466
Laminin
GCTTAGTAAAATTCTTTCTTGTCAATTTCGTTATAAGTCC


 


SEQ ID NO.: 467
IL-7
TGGCCTAATCTCGGAGACTGGCCGCTGTGGGCGCGGGCCT


 


SEQ ID NO.: 468
IL-7
GGTCAATGTCTAGTTATTAAAATATGTTTTCATAACAAAT


 


SEQ ID NO.: 469
IL-7
ATATTGTAAATACTCTTCCCTCATACAGATGATCCGGTAA


 


SEQ ID NO.: 470
IL-7
CGAGAAACCTACTTATCTTATTCTTCAATTCGATTTATTA


 


SEQ ID NO.: 471
IL-7
GCTTACCTTAACAAAATTGCAACCCAACCCTTCACCGGC


 


SEQ ID NO.: 472
IL-15
GTGACGGTGATGGTACCCGCACTGCGGCGGCGGCCAGCAG


 


SEQ ID NO.: 473
IL-15
TCTAATTATGTTACAAAATAATTGTTATGCTCCGCAAATA


 


SEQ ID NO.: 474
IL-15
CTGCCAAGTCATTACAGAATATTAAAATTTGTCATGTATT


 


SEQ ID NO.: 475
IL-15
TCATTTAATTGTCCTAAATCTGAAAATTTATTATATTTC


 


SEQ ID NO.: 476
IL-21
AAACCTGCAAAATTTAGGGCCAATGTGTGTATTGAACGGG


 


SEQ ID NO.: 477
IL-21
ATCAGAAGCTTCGATCTATTCGCCTCATTCACTCACTCTA


 


SEQ ID NO.: 478
IL-21
ACCCTATAAGCTGAGATAAGCATTCTGTGGACGAAAAGTT


 


SEQ ID NO.: 479
IL-21
GGTCGAAACAGAGAAGCCTCAAACTTAAACTTCCAATGTG


 


SEQ ID NO.: 480
IL-21
AATTTCATTCTTTAAATTGTTTTCTTAATTTTAGCTTA


 


SEQ ID NO.: 481
IL-21
TCGTATTTACCCCTATTAACATCAGATCGTGTCATAACGC


 


SEQ ID NO.: 482
IL-21
GTTATAGTAATATTGGTTCTAGCTCTCAGTAATATCAAAA


 


SEQ ID NO.: 483
IL-7R
ACGGAGATTGATTCTGTTTAAAACGGTACTATATCTTGTT


 


SEQ ID NO.: 484
IL-7R
GCACTATTTTTGACGTAACTCTTCCAATATAAAATCTGCT


 


SEQ ID NO.: 485
IL-15R
AGGCGTTAACTCTTGTCATGTTATAGACGTCTAATCCATC


 


SEQ ID NO.: 486
IL-15R
ATAGATTTTATTTTTTTTTAATTCAAATTCGCTACAGAA


 


SEQ ID NO.: 487
IL-21R
CGTTAGCGTCGTTTATACTGCAAGTACAAACTTGTAATTG


 


SEQ ID NO.: 488
IL-21R
ATGGAATATCAGCCATCGTGAATTGCTCAGACTCGAAACG


 


SEQ ID NO.: 489
IL-21R
ACTGTAATCACTTCTTTTAAATAGTCCCGGAACGATATCA


 


SEQ ID NO.: 490
IL-21R
CTTGATCCATACTATAACTTAACATTTGTTCATCTCAAGT


 


SEQ ID NO.: 491
IgG2a-
AGTTTTTGAAATGCATTACAGTATAAACATTTCACACATC



antiCD19-



 


SEQ ID NO.: 492
IgG2a-
CTAAATCGCGAACCGAGTTTTTGTCAAAGTTCTAGATTAA



antiCD19-



 


SEQ ID NO.: 493
IgG2a-
ATTTGAGAAGTTTGACTGCAGTCGCACACTCCCATTTTTG



antiCD19-



 


SEQ ID NO.: 494
IgG2a-
ATGTGTATCGATATGGCCTAACCTAGCTTTAGAACTGGTC



antiCD19-



 


SEQ ID NO.: 495
antiCD20
ATTACTTAAAGATTTGTCATCTCTTTAAAGCTTTGTTA


 


SEQ ID NO.: 496
antiCD20
CCAAAAATAGTGATCACATTTTGTGTTCGATAATAACT


 


SEQ ID NO.: 497
antiCD20
ACGAACTTTTTTAATTCGCAGACATGTTTATAGTTTCTTG


 


SEQ ID NO.: 498
antiCD20
ACGAACTTTTTTAATTCGCAGACATGTTTATAGTTTCTTG


 


SEQ ID NO.: 499
HyHel IgG
ATAGGAAGGGATTCAGCACGGGCTGTCGTAGACTTCAAGC



(chicken)



 


SEQ ID NO.: 500
LAP
AGGGTCCGCTAGACGTAGGGGAGAGCCAGAAATCTCAAC


 


SEQ ID NO.: 501
LAP
CATACCAAGTAGAGACCATACTCTCAGAGGACTGGACGCG


 


SEQ ID NO.: 502
hCG
TTTTCATTGCTACAAAGTCATTTTGTAGGTAACGGTGGAT


 


SEQ ID NO.: 503
Non gly Gly Hb
AGGGTCGTTGGGGCCAGGGTTCACGCGCCGCTCCCCGCT


 


SEQ ID NO.: 504
Non gly Gly Hb
GTGATAGGGAAGTGAGTGCTGGCCCGTAGCGACCCTGGAA


 


SEQ ID NO.: 505
Hemoglobin
GTGATAGGGAAGTGAGTGCTGGCCCGTAGCGACCCTGGAA



(glycated)



 


SEQ ID NO.: 506
Fibrinogen
GGCGCGCTGGCGCGCGAAGGTGGCTCGGAGTGCTCCGGGC



[Sigma, Cat




No. F3879]



 


SEQ ID NO.: 507
Fibronectin
CAAGAGTGTTAGACATTATCTCAGCGCTGCCAATTATATT


 


SEQ ID NO.: 508
Collagen
AGACTTGAAAGCATCTTTACTTCGATTGGTAATATTTTTGAT


 


SEQ ID NO.: 509
Laminin
TTGAAATTCAATCGCTTAAGTCCCGTTTATAGGAATAACGAT


 


SEQ ID NO.: 510
Human IL 7
TGGCCTAATCTCGGAGACTGGCCGCTGTGGGCGCGGGCCT



[PeproTech




Inc., NJ, Cat




No. 200-07]



 


SEQ ID NO.: 511
Glycated IgM
TTTTATTTCGTATCCTATATTTTCAAGTTAGCTTGACTC



(mu chain)




[IgM mu chain




from Athens




Research, and




then glycated




in-house]



 


SEQ ID NO.: 512
Non-glycated
TTTTATTTCGTATCCTATATTTTCAAGTTAGCTTGACTC



IgM mu chain




[Athens




Research]



 


SEQ ID NO.: 513
IgG Fc
TTGAAGGCGTACCGTCCGCGGGCGGCGTGTGCGCCGGGCC



[Athens




Research]



 


SEQ ID NO.: 514
Non-glycated
CTCTTAATGTCACGGCTGAGCCTATGCTGGCGTGACCGA



HSA



 


SEQ ID NO.: 515
Non-glycated
GTGTGAAAGCTGGGAGAGTCTGCGGGCCTGTGTCGCGCAA



HSA



 


SEQ ID NO.: 516
Non-glycated
ATGGGTGTGTCTGGGTAGACGTTGTTTTGGCCTGGTGTTA



HSA



 


SEQ ID NO.: 517
Glycated HSA
GTGTGAAAGCTGGGAGAGTCTGCGGGCCTGTGTCGCGCAA


 


SEQ ID NO.: 518
Glycated HSA
ATGGGTGTGTCTGGGTAGACGTTGTTTTGGCCTGGTGTTA


 


SEQ ID NO.: 519
Peroxiredoxin
CGTAAGAGGGAGATTCCTACAAACGCTCCCCATCC


 


SEQ ID NO.: 520
IL 18
CCCGCAATCCACGACACAGACGACTGCCGTGGACCACCGA


 


SEQ ID NO.: 521
Crystallin AB
CGTAGGAGGGAGTTCCAATGATACATCCTAACCGATAC


 


SEQ ID NO.: 522
Crystallin AB
GCGGGGGGTTGTGCCCCGTAAAGGCTTGCCAAGCGCCGCA


 


SEQ ID NO.: 523
Crystallin AB
GGTCAGGTACAGAAGACTGGTGTATGAAGATGCCTGCTAC


 


SEQ ID NO.: 524
RRM2
CTAATGATGGTTTTCGCAATTAACGCCATCGAACAAGATC



[Prospec,




Catalog#ENZ-




523, Swiss-




Prot# P31350]



 


SEQ ID NO.: 525
EpCAM
CCGCGCAGATATACAACGTACCTCTGTGCGCA


 


SEQ ID NO.: 526
EpCAM
CTGTGAGGCGTACTGCGGTGAGCCTCTCATTA


 


SEQ ID NO.: 527
EpCAM
CCCCCCGAATCACATGACTTGGGCGGGGGTCG


 


SEQ ID NO.: 528
EpCAM
GGCCGCGCATTCTCTGCCGGCTGGTGTACGGT


 


SEQ ID NO.: 529
EpCAM
TGACGGCCATACGTTCATCGTATGTAGTCTTC


 


SEQ ID NO.: 530
EpCAM
GGCGCAGGGGGGGGCCCAGAGTATGGGGCCTG


 


SEQ ID NO.: 531
EpCAM
CGAGGGGCGTGGGCTTCGGGCACCCAGCGGG


 


SEQ ID NO.: 532
EpCAM
ATGGCTCGGGTCTTACACCCTGGAGGACCGTG


 


SEQ ID NO.: 533
EpCAM
GGGGCGGGCACTGCCTTCGAGTTGCTCGGTGT


 


SEQ ID NO.: 534
AptaTecK TEM
CGCATGGGTACCAGTGAGCGATGGACCCTAGC


 


SEQ ID NO.: 535
Dengue2
CGCAGTGTCGTACCGTCGATGCGGGGATGCCG



protein



 


SEQ ID NO.: 536
Dengue2
CGCAGAAGGCGTCGGATAGACCCGCAATCACG



protein



 


SEQ ID NO.: 537
Hsp27
GCGGTGAACTGCTCGTAAAGCGGGGCAAGACCAGAGGGAT


 


SEQ ID NO.: 538
Hsp27
ATGCGATTGTCTCCTAATTATCACTCGCTTACTGGGTCAAT


 


SEQ ID NO.: 539
H3 K27 (Me3)
CCGATGTAGGAGTGAGGTTGGGCAGCGGGCCCAGCCGAGGCACTCCCCG



(aa 21-44)




peptide




[Anaspec, Cat.




No. 64367-025]



 


SEQ ID NO.: 540
BCM [Human
GTATCAAGCTTTAGTGGAGAGTACCACTCNCACTAAAACA



CLEC9a]



 


SEQ ID NO.: 541
BCM [Human
CCTACAGTATAGATGAGTCGACCATTAGAAACAATGGTCC



CLEC9a]



 


SEQ ID NO.: 542
Mouse CD8
GCCGTCCCCGCGTTTGGTACGCGGTAGGAGAC



[Sino




Biological




Inc., Beijing,




China [Cat:




50389-M08H]



 


SEQ ID NO.: 543
Mouse CD8
GGCGCTTAGTTAGACTAACGTTGCTAGGGGCG



[Sino




Biological




Inc., Beijing,




China [Cat No.




50389-M08H]



 


SEQ ID NO.: 544
Heparan
CTCGATCAGTACACAGATCGCCTAATGGAGATTTTTTCA



Sulfate



 


SEQ ID NO.: 545
Epirubicin
CACTGGGGTCGGAGATTTCTCGTTGTGGCGGCCGCCGGCG


 


SEQ ID NO.: 546
Ampicillin
ATTAATATCTAACTAGCGCGCTCGTCTCAATATCGGCAAG


 


SEQ ID NO.: 547
Tetracycline
GTTTGTGTATTACAGTTATGTTACCCTCATTTTTCTGAAC









It will be appreciated by those of ordinary skill in the art that the present invention can be embodied in other specific forms without departing from the spirit or essential character hereof. The present description is therefore considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the appended claims, and all changes that come within the meaning and range of equivalents thereof are intended to be embraced therein.

## Claims

1. A ligand to a target molecule comprising a nucleic acid sequence having at least 95% homology or identity to a sequence selected from the group consisting of sequence IDs Seq40, Seq41, Seq42, Seq44, Seq45, Seq46, Seq47, Seq48, and Seq49.

2. A ligand to a target molecule comprising a modified nucleic acid sequence having at least 95% homology or identity to a sequence selected from the group consisting of sequence IDs Seq40, Seq41, Seq42, , Seq44, Seq45, Seq46, Seq47, Seq48, and Seq49.