Systematic evolution of ligands by exponential enrichment: Solution SELEX

• US 5,567,588 A
• Filed: 06/05/1995
• Issued: 10/22/1996
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: University of Colorado System
• Inventors: Ringquist, Steven, Gold, Larry
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/461,069
• Time in Patent Office: 505 Days
• Field of Search: 435/6, 435/91.2, 435/77, 435/78
• US Class Current: 435/6.11
• CPC Class Codes: A61K 47/547 Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/13 Decoys
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 four
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/575 : Hormones
• CPC Class Codes: G01N 2333/62 : Insulins
• CPC Class Codes: G01N 2333/8125 : Alpha-1-antitrypsin
• CPC Class Codes: G01N 2333/96433 : Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 2333/96436 : Granzymes
• CPC Class Codes: G01N 2333/96455 : Kallikrein (3.4.21.34; 3.4.21.35)
• CPC Class Codes: G01N 2333/96486 : Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 2333/966 : Elastase
• CPC Class Codes: G01N 2333/9726 : Tissue plasminogen activator
• CPC Class Codes: G01N 2333/974 : Thrombin
• CPC Class Codes: G01N 2333/976 : Trypsin; Chymotrypsin
• CPC Class Codes: G01N 33/531 : Production of immunochemical test materials
• CPC Class Codes: G01N 33/532 : Production of labelled immunochemicals
• CPC Class Codes: G01N 33/535 : with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

Described herein are methods for improved partitioning between high and low affinity nucleic acid ligands identified through the SELEX method, termed solution SELEX. The solution SELEX method achieves partitioning between high and low affinity nucleic acid-target complexes through a number of methods, including (1) primer extension inhibition which results in differentiable cDNA products. Primer extension inhibition is achieved with the use of nucleic acid polymerases, including DNA or RNA polymerases, reverse transcriptase, and Qβ-replicase; (2) exonuclease hydrolysis inhibition which results in only the highest affinity ligands amplifying during PCR. This is achieved with the use of any 3'"'"'→5'"'"' double-stranded exonuclease; (3) linear to circle formation to generate molecules amplifiable during PCR; or (4) PCR amplification of single-stranded nucleic acids. A central theme of the method of the present invention is that the nucleic acid candidate mixture is screened in solution and results in preferential amplification of the highest affinity RNA ligand or catalytic RNA.

Claims

1. A method for identifying nucleic acid ligands from a candidate mixture of nucleic acids, said nucleic acid ligands being a ligand of a given target molecule comprising:

a) contacting said candidate mixture with the target molecule, wherein nucleic acids having increased affinity to the target molecule form nucleic acid-target complexes;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, said partitioning step resulting in two differentiable nucleic acid pools said nucleic acids in each pool having different relative affinities to said target molecule; and

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule may be identified.

2. The method of claim 1 wherein said partitioning in step b) comprises:

(i) performing a first primer extension with a nucleic acid polymerase under conditions wherein the increased affinity nucleic acids associated with said nucleic acid-target complexes are not primed;

(ii) removing the target molecule;

(iii) performing a second primer extension with a nucleic acid polymerase under conditions different from step (i), wherein said increased affinity nucleic acids are primed and said two differentiable nucleic acid, pools result from step (i) and step (iii); and

(iv) partitioning the increased affinity nucleic acid pool of step (iii) from the remainder of the candidate mixture.

3. The method of claim 2, wherein said nucleic acid polymerase is selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, and Qβ

-replicase.

4. The method of claim 2 wherein said first primer extension is performed in the presence of chain terminating nucleotides and said second primer extension is performed in the absence of chain terminating nucleotides, wherein only the primer extension product from the increased affinity oligonucleotide is amplifiable by a polymerase chain reaction.

5. The method of claim 2 wherein said first primer extension is performed in the presence of unmodified nucleotides, followed by removal of said target, said second primer extension is performed in the presence of modified nucleotides resistant to enzymatic cleavage by single- or double-stranded nucleases, or uracil DNA glycosylase, and the primer extension products are incubated with an appropriate cleaving enzyme, whereby only the primer extension product from the increased affinity oligonuclcotides are amplifiable by a polymerase chain reaction.

6. The method of claim 2 wherein said first primer extension is performed in the presence of modified nucleotides that allow retention of the first primer extension product on an affinity matrix, followed by removal of said target, said second primer extension is performed in the presence of unmodified nucleotides, and the product primed from said first primer extension is removed by retention on an affinity matrix.

7. The method of claim 2 wherein said first primer extension is performed in the presence of modified nucleotides, followed by removal of said target, said second primer extension is performed in the presence of modified nucleotides that allow retention of the second primer extension product on an affinity matrix, and the product of the second primer extension from increased affinity oligonucleotides is removed by retention on an affinity matrix.

10. The method of claim 5 wherein said cleaving enzyme is selected from the group consisting of restriction endonuclease and uracil DNA glycosylase.

8. A method for identifying nucleic acid ligands from a candidate mixture of nucleic acids, said nucleic acid ligands being a ligand of a given target molecule comprising:

a) contacting said candidate mixture with the given target molecule, wherein nucleic acids having an increased affinity to the target molecule form nucleic acid-target complexes;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, said partitioning step comprising;

(i) performing a first primer extension with a nucleic acid polymerase in the presence of nucleotides sensitive to chemical cleavage;

(ii) removing the target molecule;

(iii) performing a second primer extension with a nucleic acid polymerase in the presence of modified nucleotides resistant to chemical cleavage;

(iv) incubating said first and second primer extension products with a nucleotide cleaving chemical, whereby the extension product of the first primer extension is cleaved; and

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the given target molecule may be identified.

9. A method for identifying double-stranded nucleic acid ligands from a candidate mixture of nucleic acids, said double-stranded nucleic acid ligands being a ligand of a given target molecule comprising:

a) contacting said candidate mixture with the given target molecule, wherein nucleic acids having an increased affinity to the target molecule form nucleic acid-target complexes;

b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture, said partitioning step comprising;

(i) incubating the increased affinity nucleic acids with exonuclease wherein full length double-stranded nucleic acids not forming nucleic acid-target complexes are degraded and the double-stranded nucleic acids forming nucleic acid-target complexes are partially protected from degradation;

(ii) removing said exonuclease and said target;

(iii) primer extending said double-stranded nucleic acids with polymerase, wherein the double-stranded nucleic acid ligand-enriched candidate mixture is regenerated from the partially protected nucleic acids; and

c) amplifying the primer extended double-stranded nucleic acids to yield a ligand-enriched mixture of double-stranded nucleic acids, whereby the double-stranded nucleic acid ligands of the given target molecule may be identified.