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WO2003014369A1 - Ligands d'acides nucleiques a duplexes intramoleculaires - Google Patents

Ligands d'acides nucleiques a duplexes intramoleculaires Download PDF

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Publication number
WO2003014369A1
WO2003014369A1 PCT/US2002/027085 US0227085W WO03014369A1 WO 2003014369 A1 WO2003014369 A1 WO 2003014369A1 US 0227085 W US0227085 W US 0227085W WO 03014369 A1 WO03014369 A1 WO 03014369A1
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Prior art keywords
nucleic acid
target
ligand
candidate mixture
target molecule
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PCT/US2002/027085
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English (en)
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Larry Gold
Edward N. Brody
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Somalogic, Inc.
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Publication of WO2003014369A1 publication Critical patent/WO2003014369A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • This invention is directed to a method for the generation of nucleic acid ligands having specific functions against target molecules using the SELEX process.
  • SELEX process Systematic Evolution of Ligands by Exponential enrichment
  • nucleic acids have three dimensional structural diversity not unlike proteins.
  • the SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in United States Patent Application Serial No. 07/536,428, filed June 11, 1990, entitled “Systematic Evolution of Ligands by Exponential Enrichment," now abandoned, United States Patent No. 5,475,096 entitled “Nucleic Acid Ligands", United States Patent No.
  • the SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • the SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
  • the present inventors have recognized that SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target.
  • SELEX or SELEX-like processes could be used to identify nucleic acids which can facilitate any chosen reaction in a manner similar to that in which nucleic acid ligands can be identified for any given target.
  • the present inventors postulate that at least one nucleic acid exists with the appropriate shape to facilitate each of a broad variety of physical and chemical interactions.
  • the resulting nucleic acid ligands are often referred to as "photoaptamers.” These patents and patent applications are referred to in this application collectively as “the photoSELEX process applications.” In the photoSELEX process variation of the SELEX process, a modified nucleotide activated by absorption of light is incorporated in place of a native base in either RNA- or in ssDNA-randomized oligonucleotide libraries.
  • United States Patent No. 5,580,737 entitled “High- Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. United States Patent No.
  • the SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in United States Patent No. 5,660,985 entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides," that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. United States Patent No.
  • the SELEX process has been adapted in order to allow the gh-throughput, automated generation of high affinity nucleic acid ligands to targets of interest.
  • Methods and apparatus for automated generation of nucleic acid ligands are described in United States Patent Application Serial No. 09/232,946, filed January 19, 1999, United States Patent Application Serial No. 09/356,233 filed July 16, 1999, United States Patent Application Serial No. 09/616,284, filed July 14, 2000, and United States Patent Application Serial No. 09/815,171, filed March 22, 2001, each of which is entitled “Methods and Apparatus for the Automated Generation of Nucleic Acid Ligands.” These patent applications are collectively referred to as "the automated SELEX process applications.”
  • nucleic acid ligand can be made to increase the in vivo stability of the nucleic acid ligand or to enhance or to mediate the delivery of the nucleic acid ligand. See, e.g., U.S. Patent Application Serial No. 08/117,991, filed September 9, 1993, now abandoned, and United States Patent No.
  • nucleic acid ligands contemplated in these applications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
  • the nucleic acid ligands are DNA molecules that are modified with a photoreactive group on the 5-position of pyrimidine residues.
  • the modifications can be pre- or post-SELEX process modifications.
  • Nucleic acid ligands may be attached to planar solid supports to form microarrays.
  • microarrays also commonly referred to as “biochips”
  • methods for their manufacture and use are described in United States Patent Application Serial No. 08/990,436, filed December 15, 1997, United States Patent Application Serial No. 08/211,680, filed December 14, 1998, now abandoned, Patent Cooperation Treaty Application Serial No.
  • nucleic acid ligands of targets implicated in disease are attached to a planar solid support in an array format, and the solid support is then contacted with a bodily fluid suspected of containing those targets. Quantitation of the level of target binding to each nucleic acid ligand is then used to provide diagnostic or prognostic medical information.
  • each nucleic acid ligand in the array is generated using the photoSELEX process as detailed in the photoSELEX process applications.
  • nucleic acid ligands After such nucleic acid ligands have been contacted with the fluid to be assayed, they are photoactivated and the solid support is washed under very stringent conditions (preferably under conditions that denature nucleic acids) in order to remove non-specifically bound molecules; bound target is not removed because it is covalently crosslinked to nucleic acid ligand via the photoreactive group.
  • target quantitation can then be achieved by using a reagent that labels all proteins with a detectable group, such as a fluorescent group.
  • Nucleic acid ligands can be attached to solid supports by a number of methods well known in the art, and detailed in the biochip applications.
  • the 5' or 3' terminus of the nucleic acid ligand is covalently-coupled to a derivatized solid support.
  • nucleic acid ligands in such diagnostic applications are that occasionally a nucleic acid ligand might non-specifically bind molecules other than its cognate target contained in bodily fluids. It is believed that single-stranded nucleic acid has the potential to be bound by certain proteins, such as heparin-binding proteins (including Platelet
  • PDGF Derived Growth Factor
  • a protein that non-specifically binds single-stranded nucleic acid may be sufficiently close to a photoreactive group to become photocrosslinked to the nucleic acid ligand. If this occurs, then even stringent washing of the solid support will not remove the non-specifically bound protein, and quantitation of target binding to the nucleic acid ligand will be inaccurate. Although these problems are expected to occur very infrequently, it would be desirable nonetheless to have a method for obtaining nucleic acid ligands that do not have single stranded regions at their 5' or 3' ends.
  • the 5' and/or the 3' terminus of the nucleic acid ligand may be bound to a solid support to form a spatially-localized nucleic acid ligand.
  • a plurality of such bound nucleic acids on a solid support constitutes a nucleic acid ligand array or biochip.
  • the length of the intramolecular duplex can be chosen such that the distance between the target-binding portion of the nucleic acid ligand and the solid support is optimal for target binding.
  • the invention also includes methods and reagents for generating nucleic acid ligands of composition 5' A-L-A' 3' by the SELEX process.
  • the invention includes a method for the identification of a nucleic acid ligand from a candidate mixture of nucleic acids, each said nucleic acid in said candidate mixture comprising the sequence 5' A-R-A 3', wherein R is at least partially-randomized sequence, and wherein A and A' are fixed sequence regions complementary in sequence to one another that are capable of forming an intramolecular duplex, the method comprising the steps: a) contacting the candidate mixture with the target wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids; wherein a nucleic acid ligand is identified in which A and A' are capable of forming an intramolecular duplex.
  • the invention also provides photocrosslinking nucleic acid ligands of the general composition 5' A-L-A' 3' , wherein the target-binding region L comprises one or more photoreactive groups, and wherein A and A' are flanking mutually-complementary sequences that can base-pair with one another to form an intramolecular duplex.
  • the 5' and/or the 3' tenninus of the nucleic acid ligand may be bound to a solid support to form a spatially- localized photocrosslinking nucleic acid ligand.
  • a plurality of such bound photocrosslinking nucleic acids on a solid support constitutes a nucleic acid ligand array or biochip.
  • the photocrosslinking nucleic acid ligands of the instant invention are generated by the process of identifying a nucleic acid ligand of a target from a candidate mixture of nucleic acids comprising the sequence 5' A-R-A' 3', wherein R is at least partially-randomized sequence comprising one or more photoreactive groups, said process comprising: a) contacting said candidate mixture with said target molecule, wherein nucleic acid sequences having increased affinity to the target molecule relative to the candidate mixture form nucleic acid-target molecule complexes; b) irradiating said candidate mixture, wherein said nucleic acid-target molecule complexes photocrosslirik; c) partitioning the crosslinked nucleic acid-target molecule complexes from free nucleic acids in the candidate mixture; and d) identifying the nucleic acid sequences that photocrosslinked to the target molecule.
  • Figure 1 illustrates schematically the attachment to a solid surface 102 of a nucleic acid
  • composition 5' A-L-A' 3' wherein L comprises the target-binding portion of the nucleic acid ligand, wherein A and A' are flanking mutually-complementary sequences that can base- pair with one another to form an intramolecular duplex, and wherein A is attached to a solid support.
  • SELEX process an acronym for Systematic Evolution of Ligands by Exponential enrichment.
  • nucleic acid ligand is a non-naturally occurring nucleic acid having a desirable action on a target.
  • Nucleic acid ligands are often referred to as “aptamers”.
  • the term aptamer is used interchangeably with nucleic acid ligand throughout this application.
  • a desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, facilitating the reaction between the target and another molecule.
  • the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to the nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
  • Nucleic acid ligands include nucleic acids that are identified from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of a given target, by the method comprising: a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids.
  • candidate mixture is a mixture of nucleic acids comprised of differing regions of sequence from which to select a desired ligand.
  • the source of a candidate mixture can be from naturally-occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the foregoing techniques.
  • each nucleic acid in a candidate mixture comprises an at least-partially randomized sequence flanked on either side by mutually complementary fixed sequences that can form an intramolecular duplex.
  • each nucleic acid has additional fixed sequences on either side of an at least-partially randomized sequence region to facilitate the amplification process.
  • fixed terminal "tail" sequences are added to the fixed sequence regions and primers used for amplification in order to prevent the formation of high molecular weight "parasites" of the amplification process, which are believed to result from rare mispriming events.
  • Such "tails” obviate the need to size fractionate amplification products at the conclusion of each round of the SELEX process or the photoSELEX process, and are therefore especially useful in the automated SELEX process and the automated photoSELEX process.
  • nucleic acid means either DNA, RNA, single-stranded or double- stranded, and any chemical modifications thereof. Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
  • Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, methylations, unusual base- pairing combinations such as the isobases isocytidine and isoguanidine and the like.
  • An example of a backbone modified nucleic acid included in the instant invention is peptide nucleic acid (PNA). Modifications can also include 3' and 5' modifications such as capping.
  • SELEX methodology involves the combination of selection of nucleic acid ligands which interact with a target in a desirable manner, for example binding to a protein, with amplification of those selected nucleic acids. Optional iterative cycling of the selection/amplification steps allows selection of one or a small number of nucleic acids which interact most favorably with the target from a pool which contains a very large number of nucleic acids. Cycling of the selection/amplification procedure is continued until a selected goal is achieved.
  • the SELEX methodology is described in the SELEX Patent Applications. "SELEX target” or “target molecule” or “target” refers herein to any compound upon which a nucleic acid can act in a predetermined desirable manner.
  • a SELEX target molecule can be a protein, peptide, nucleic acid, carbohydrate, lipid, polys accharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell, tissue, etc., without limitation. Virtually any chemical or biological effector would be a suitable SELEX target. Molecules of any size can serve as SELEX targets. A target can also be modified in certain ways to enhance the likelihood of an interaction between the target and the nucleic acid.
  • tissue target or “tissue” refers herein to a certain subset of the SELEX targets described above.
  • tissues are macromolecules in a heterogeneous environment.
  • tissue refers to a single cell type, a collection of cell types, an aggregate of cells, or an aggregate of macromolecules. This differs from simpler SELEX targets which are typically isolated soluble molecules, such as proteins.
  • tissues are insoluble macromolecules which are orders of magnitude larger than simpler SELEX targets.
  • Tissues are complex targets made up of numerous macromolecules, each macromolecule having numerous potential epitopes. The different macromolecules which comprise the numerous epitopes can be proteins, lipids, carbohydrates, etc., or combinations thereof.
  • Tissues are generally a physical array of macromolecules that can be either fluid or rigid, both in terms of structure and composition. Extracellular matrix is an example of a more rigid tissue, both structurally and compositionally, while a membrane bilayer is more fluid in structure and composition. Tissues are generally not soluble and remain in solid phase, and thus partitioning can be accomplished relatively easily. Tissue includes, but is not limited to, an aggregate of cells usually of a particular kind together with their intercellular substance that form one of the structural materials commonly used to denote the general cellular fabric of a given organ, e.g., kidney tissue, brain tissue.
  • the four general classes of tissues are epithelial tissue, connective tissue, nerve tissue and muscle tissue.
  • tissue which fall within this definition include, but are not limited to, heterogeneous aggregates of macromolecule such as fibrin clots which are acellular; homogeneous or heterogeneous aggregates of cells; higher ordered structures containing cells which have a specific function, such as organs, tumors, lymph nodes, arteries, etc.; and individual cells.
  • Tissues or cells can be in their natural environment, isolated, or in tissue culture. The tissue can be intact or modified. The modification can include numerous changes such as transformation, transfection, activation, and substructure isolation, e.g., cell membranes, cell nuclei, cell organelles, etc.
  • Sources of the tissue, cell or subcellular structures can be obtained from prokaryotes as well as eukaryotes. This includes human, animal, plant, bacterial, fungal and viral structures.
  • solid support is defined as any surface to which molecules may be attached through either covalent or non-covalent bonds. This includes, but is not limited to, membranes, microtiter plates, magnetic beads, charged paper, nylon, Langmuir-Bodgett films, functionalized glass, germamum, silicon, PTFE, polystyrene, gallium arsenide, gold, and silver. Any other material known in the art that is capable of having functional groups such as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also contemplated. This includes surfaces with any topology, including, but not limited to, spherical surfaces and grooved surfaces.
  • biological fluid refers to any biological substance, including but not limited to, blood (including whole blood, leukocytes prepared by lysis of red blood cells, peripheral blood mononuclear cells, plasma, and serum), sputum, urine, semen, cerebrospinal fluid, bronchial aspirate, sweat, feces, synovial fluid, macerated tissue, and tissue extracts.
  • biological fluid typically contains cells and their associated molecules, soluble factors, small molecules and other substances.
  • photoaptamers refers to nucleic acid ligands that contain a photoreactive group that can crosslink with the target.
  • Suitable photoreactive groups include, but are not limited to, 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5- azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5- iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8- azidoguanine, 8-bromoguar ⁇ ne, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bro
  • nucleic acid ligands of the present invention are derived from the SELEX process methodology.
  • the SELEX process is described in United States Patent Application Serial No. 07/536,428, entitled “Systematic Evolution of Ligands by Exponential Enrichment,” now abandoned, United States Patent No. 5,475,096 entitled “Nucleic Acid Ligands,” and in United States Patent No. 5,270,163 (see also, WO 91/19813) entitled “Nucleic Acid Ligands.”
  • These applications, each specifically incorporated herein by reference, are collectively called the SELEX Patent Applications.
  • the SELEX process provides a class of products which are nucleic acid molecules, each having a unique sequence, and each of which has the property of binding specifically to a desired target compound or molecule.
  • Target molecules are preferably proteins, but can also include among others carbohydrates, peptidoglycans and a variety of small molecules.
  • SELEX methodology can also be used to target biological structures, such as cell surfaces or viruses, through specific interaction with a molecule that is an integral part of that biological structure.
  • the SELEX process may be defined by the following series of steps: 1) A candidate mixture of nucleic acids of differing sequence is prepared.
  • the candidate mixture generally includes regions of fixed sequences (i.e., each of the members of the candidate mixture contains the same sequences in the same location) and regions of randomized sequences.
  • the fixed sequence regions are chosen either: (a) to assist in the amplification steps described below, (b) to mimic a sequence known to bind to the target, (c) to enhance the concentration of a given structural arrangement of the nucleic acids in the candidate mixture, and/or (d) to function as "tails" that prevent high molecular weight parasites of the amplification process from forming in the absence of size fractionation of amplifications products, as described above.
  • the randomized sequences can be totally randomized (i.e., the probability of findmg a base at any position being one in four) or only partially randomized (e.g., the probability of finding a base at any location can be selected at any level between 0 and 100 percent).
  • the candidate mixture is contacted with the selected target under conditions favorable for binding between the target and members of the candidate mixture. Under these circumstances, the interaction between the target and the nucleic acids of the candidate mixture can be considered as forming nucleic acid-target pairs between the target and those nucleic acids having the strongest affinity for the target.
  • nucleic acids with the highest affinity for the target are partitioned from those nucleic acids with lesser affinity to the target. Because only an extremely small number of sequences (and possibly only one molecule of nucleic acid) corresponding to the highest affinity nucleic acids exist in the candidate mixture, it is generally desirable to set the partitioning criteria so that a significant amount of the nucleic acids in the candidate mixture
  • nucleic acids selected during partitioning as having the relatively higher affinity for the target are then amplified to create a new candidate mixture that is enriched in nucleic acids having a relatively higher affinity for the target.
  • the newly formed candidate mixture contains fewer and fewer unique sequences, and the average degree of affinity of the nucleic acids to the target will generally increase. Taken to its extreme, the
  • SELEX process will yield a candidate mixture containing one or a small number of unique nucleic acids representing those nucleic acids from the original candidate mixture having the highest affinity to the target molecule.
  • the candidate nucleic acid mixture comprises fixed sequence regions that are complementary to one another.
  • a candidate nucleic acid mixture can be prepared in which an at least-partially random sequence region is flanked 5' by the fixed sequence:
  • each nucleic acid in the candidate mixture will therefore comprise an intramolecular duplex "stem" formed by the complementary fixed regions, and a region comprising the at least partially-randomized random sequence that connects the strands of the "stem".
  • the exact structure of the at least partially-randomized region of each nucleic acid in the candidate mixture, and hence its propensity for target binding, will depend on the sequence of the random region.
  • intramolecular duplex candidate mixture has the general composition 5' A-R-A' 3', wherein R is the at least-partially randomized sequence region, and A and A' are the complementary fixed sequence regions.
  • the intramolecular duplex candidate mixture can be used to perform the SELEX process as described in the SELEX patent applications. This leads to the generation of nucleic acid ligands of general composition 5' A-L-A' 3' wherein L comprises the target-binding portion of the nucleic acid ligand.
  • the intramolecular candidate mixture is amplified during the SELEX process by the Polymerase Chain Reaction (PCR) with primers complementary in sequence to A and A'.
  • the PCR procedure is preferably carried out with a concentration of primer sufficiently high to insure that fixed regions of each nucleic acid in the intramolecular duplex candidate mixture are more likely to anneal to primer than to one another.
  • the candidate mixture comprises additional fixed sequence regions 5' of A and 3' of A'.
  • Such additional fixed sequence regions serve as primer annealing sites for the amplification of the intramolecular duplex candidate mixture.
  • Such candidate mixture can be represented as 5' P ⁇ -A-R-A'-P 2 3', wherein P 1 and P 2 are the fixed sequence regions used for PCR amplification, R is the at, least-partially randomized region, and A and A' form the intramolecular duplex.
  • the fixed sequence regions Pi and P 2 used for amplification have higher Tm's than the fixed sequence regions A and A' that form the intramolecular duplex.
  • nucleic acids ligands When the SELEX process is performed according to the abovementioned embodiments with an intramolecular duplex candidate mixture, nucleic acids ligands will be obtained in which the target binding by the L region can take place in the context of an intramolecular duplex formed between the complementary fixed sequence regions A and A'.
  • the L region can adopt its target-binding conformation when (and in some cases, only if) the flanking complementary fixed regions A and A' of the nucleic acid ligand form an intramolecular duplex.
  • the nucleic acid ligand thus identified can be attached, either covalently or non-covalently, to a solid support via the A- A' intramolecular duplex.
  • the 5' end of A and/or the 3' end of A' can be attached to the solid support. Any suitable technique known in the art for the attachment of nucleic acids to solid supports is contemplated by the instant invention.
  • FIGURE 1 illustrates the attachment of a nucleic acid ligand of the instant invention 101 via its 5' terminus to a solid support 102.
  • target binding region L is shown schematically as a loop. The exact conformation of region L in any given nucleic acid ligand will depend upon its sequence, and may comprise one or more intramolecular duplexes.
  • the nucleic acid ligands in embodiments in which additional fixed sequences (such as Pi and P above) are present 5' and 3' of the complementary fixed sequences A and A', the nucleic acid ligands, once identified, can be synthesized without the additional fixed sequences by standard nucleic acid synthesis techniques.
  • the intramolecular candidate mixture is 5' Pi-A-R-A'-P 2 3'
  • the nucleic acid ligands identified can be represented as 5' Pi-A- L-A'-P 2 3'.
  • the identified nucleic acid ligands can then be synthesized lacking the sequences Pi and P 2 . This allows the nucleic acid ligand to be attached to a solid support via the intramolecular duplex between A and A'.
  • duplex nucleic acid has a defined, rigid structure.
  • the intramolecular duplex formed by A and A' when anchored to a solid support, can thus be thought of as a "scaffold" upon which the target-binding L region the nucleic acid ligand is displayed. Given the comparative rigidity and uniformity of the intramolecular duplex, target binding will be more efficient and reproducible than if attachment occurs through single-stranded nucleic acid.
  • the nucleic acid ligands of the instant invention are less likely to bind proteins non-specifically than are nucleic acid ligands that have single-stranded 5' and 3' regions.
  • the length of the A- A' intramolecular duplex, and hence the distance between the target-binding L region of the nucleic acid ligand and the solid support, is selected to allow for optimal target binding.
  • the A- A' duplex can be either truncated or extended relative to the original candidate mixture by deleting or adding complementary nucleotides respectively to A and A' during synthesis. Because the A-A' duplex merely provides a scaffold upon which the target-binding L region of the nucleic acid ligand is displayed, the exact sequence of the duplex is unimportant.
  • the methods and reagents of the instant invention are used to perform the photoSELEX process. The photoSELEX process is described in great detail in the photoSELEX process applications mentioned above. Once identified by the photoSELEX process, nucleic acids ligands can then be synthesized such that photoreactive nucleotides are incorporated only into the L region.
  • nucleic acid ligands sometimes are degraded in bodily fluids by exonucleases and endonucleases. Nucleic acid ligands identified according to the methods of the instant invention can synthesized with peptide linkages (instead of phosphodiester linkages) between the nucleotides or ribonucleotides that form each strand of the intramolecular duplex.
  • Nucleic acid with such peptide linkages is referred to as peptide nucleic acid (PNA) and is well known in the art.
  • PNA peptide nucleic acid
  • the three nucleotides or ribonucleotides at the 3' terminus and the three nucleotides or ribonucleotides at the 5' terminus are synthesized with peptide linkages.
  • the presence of PNA at the terminus of the intramolecular duplex prevents the digestion of the nucleic acid ligand at this location by exonucleases and endonucleases as these enzymes are unable to cleave peptide linkages.
  • the SELEX process or the photoSELEX process can be automated to generate nucleic acid ligands with little or no operator intervention.
  • the automated SELEX process method involves: a) contacting a candidate mixture of nucleic acid ligands in a containment vessel with a target molecule that is associated with a solid support; b) incubating the candidate mixture and the solid support in the containment vessel at a predetermined temperature to allow candidate nucleic acid ligands to interact with the target; c) partitioning the solid support with bound target and associated nucleic acid ligands away from the candidate mixture; d) optionally washing the solid support under predetermined conditions to remove nucleic acid that are associated non-specifically with the solid support or the containment vessel; e) releasing from the solid support the nucleic acid ligands that interact specifically with the target; f) amplifying, purifying and quantifying the released nucleic acid ligands; g
  • the automated photoSELEX process involves the steps: a) contacting a candidate mixture of nucleic acids with a target, each nucleic acid comprising one or more photoreactive groups, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture form nucleic acid-target complexes with the target; b) irradiating said complexes, wherein said nucleic acid-target complexes photocrosslink; c) partitioning the photocrosslinked nucleic acid-target complexes from the remainder of said candidate mixture; and d) identifying a nucleic acid ligand that photocrosslinked to the target; wherein steps a)-c) are performed automatically by the computer-controlled robotic manipulator.
  • the intramolecular duplex candidate mixtures of the instant invention be used to perform the automated SELEX process and the automated photoSELEX process.
  • the methods of the instant invention will greatly facilitate the efficient generation of large number of nucleic acid ligands that can be attached to solid supports in a reproducible and uniform manner.
  • the dominant nucleic acid product occasionally comprises high molecular weight nucleic acids without ligand activity. While not wishing to be bound by any particular theory, it is believed that these nucleic acid species—which we term "parasites"— result from rare mispriming events that occur during PCR. These mispriming events are believed to occur when rare candidate nucleic acid ligands contain a sequence in their random regions that is complementary in sequence to the 3' fixed sequence used for PCR amplification. If the 3' fixed sequence folds back over this complementary sequence in the random region, a self-priming intramolecular duplex may form. This structure can extended by Taq polymerase to form a longer product during PCR amplification.
  • the 3' fixed sequence of another candidate nucleic acid ligand can form an intermolecular duplex with the complementary sequence in the random region, and the 3' end of the former candidate nucleic acid can be extended by Taq polymerase to form a longer product.
  • a series of either of the events will produce parasites with a variable number of repeats. Once these parasites have formed, they will anneal promiscuously with other nucleic acids, including the correct products, leading to the formation of ever-larger parasites through 3' end extension. As parasites grow, they contain more and more primer binding sites, allowing them to be efficiently amplified during the PCR process at the expense of bona fide nucleic acid ligands. In the most extreme cases, nucleic acid ligand products are sometimes eliminated from the candidate mixture of nucleic acid ligands that contains a parasite.
  • PCR can be performed with one primer linked to a tail sequence ATATATAT , and the other linked to the tail sequence TTTTTTTT.
  • the correct PCR product will have ATATATAT on the 3' terminus of one strand and AAAAAAAA on the 3' terminus of the other strand.
  • the tail sequences AAAAAAAA and ATATATAT will not anneal intra- or intermolecularly to the random regions of candidate nucleic acid ligands that fortuitously contain just those sequences. It will be recognized by those skilled in the art that other sequences with low Tm may also be used.
  • the 5' ends of the primers used for PCR amplification of the intramolecular duplex candidate mixture are attached to tail sequences selected according to the abovementioned criteria, hi addition, the intramolecular duplex candidate mixture used to initiate the automated SELEX process also preferably has tail sequences attached to both the 5' and 3' termini. Each tail sequence is chosen to have a lower
  • Tm than the any of the fixed sequence regions in the candidate mixture (e.g., lower than the
  • a suitable intramolecular duplex candidate mixture can be represented by the formula 5' T 1 -P 1 -A-R-A'-P 2 - T 2 3', wherein Ti and T 2 are tail sequences, Pi and P 2 are fixed sequence regions used for PCR amplification, A and A' are complementary fixed sequences that are capable of forming the intramolecular duplex, and R is the at least partially-randomized sequence region.
  • the nucleic acid ligand can be synthesized without the tail sequences or the primer annealing sequences used for amplification, hi embodiments of the invention in which the automated photoSELEX process is performed with tailed primers and a tailed candidate mixture, the tail sequences will preferably not contain any photoreactive groups.

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Abstract

La présente invention concerne des méthodes ainsi que des réactifs conçus pour obtenir des ligands d'acides nucléiques par le processus SELEX. En particulier, la présente invention concerne des méthodes et des réactifs conçus pour obtenir des ligands d'acides nucléiques de composition générale 5' A-L-A' 3', dans laquelle L forme la région de liaison cible du ligand d'acides nucléiques, et dans laquelle la séquence A est complémentaire en séquence à la séquence A', de sorte que A et A' soient capables de former un duplexe intramoléculaire. Lesdits ligands d'acides nucléiques de l'invention sont obtenus par exécution du processus SELEX ou du processus PHOTOSELEX au moyen d'un mélange candidat d'acides nucléiques, chaque acide nucléique dans le mélange candidat comprenant la séquence 5' A-R-A' 3', dans laquelle R est une région d'au moins une séquence partiellement aléatoire.
PCT/US2002/027085 2001-08-09 2002-08-08 Ligands d'acides nucleiques a duplexes intramoleculaires WO2003014369A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005022153A3 (fr) * 2003-02-19 2005-06-23 Syntherica Corp Compositions et procedes de criblage utilisant des populations d'anticorps de substitution
US8409795B2 (en) 2007-07-17 2013-04-02 Somalogic, Inc. Selex and photoSELEX

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001577A (en) * 1998-06-08 1999-12-14 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
JP2001103975A (ja) * 1999-10-08 2001-04-17 Agency Of Ind Science & Technol モジュレートアプタマー及びこれを用いた標的タンパク質の検出方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001577A (en) * 1998-06-08 1999-12-14 Nexstar Pharmaceuticals, Inc. Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
JP2001103975A (ja) * 1999-10-08 2001-04-17 Agency Of Ind Science & Technol モジュレートアプタマー及びこれを用いた標的タンパク質の検出方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005022153A3 (fr) * 2003-02-19 2005-06-23 Syntherica Corp Compositions et procedes de criblage utilisant des populations d'anticorps de substitution
US8409795B2 (en) 2007-07-17 2013-04-02 Somalogic, Inc. Selex and photoSELEX

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