Parallel selex

• US 5,858,660 A
• Filed: 03/20/1996
• Issued: 01/12/1999
• Est. Priority Date: 09/20/1994
• Status: Expired due to Term

• Current Assignee: EP Technology (Boston Scientific Corporation), Medallion Biomedical, LLC
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Eaton, Bruce
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/618,700
• Time in Patent Office: 1,028 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/25.4, 935/77, 935/78
• US Class Current: 506/32
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/205 Aptamer
• CPC Class Codes: C12Q 2541/101 Selex
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: F02B 2075/027 : four
• CPC Class Codes: G01N 2333/16 : HIV-1, HIV-2
• CPC Class Codes: G01N 2333/163 : Regulatory proteins, e.g. tat, nef, rev, vif, vpu, vpr, vpt, vpx
• CPC Class Codes: G01N 2333/503 : basic FGF [bFGF]
• 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 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/76 : Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors

Abstract

This invention discloses a method for coevolving products from two or more reactants, along with the nucleic acid that can facilitate the reaction for making the products. The invention further discloses the products and facilitating nucleic acids produced by said method.

Claims

1. A method for producing a product having the ability to perform a preselected function on a target comprising:

(a) preparing a nucleic acid test mixture;

(b) coupling each member of said nucleic acid test mixture with a first reactant consisting of a small organic molecule with a molecular weight in the range of 2 to 1000 to form a nucleic acid-first reactant test mixture;

(c) forming a product library by contacting said nucleic acid-first reactant test mixture with a mixture of free reactants consisting of small organic molecules with a molecular weight in the range of 2 to 1000, wherein said product library is formed as a result of a bond formation reaction between said first reactant and at least one of said free reactants, wherein said bond formation reaction is facilitated by a nucleic acid coupled to said first reactant;

(d) contacting the product library of step (c) with a target, wherein the product having the ability to perform a preselected function on said target relative to the product library may be partitioned from the remainder of the product library; and

(e) partitioning said product having said ability to perform a preselected function on said target from the remainder of the product library, whereby said product can be identified.

5. The method of claim 1 wherein said nucleic acid test mixture comprises nucleic acids having a region of conserved sequences and a region of randomized sequences.

6. The method of claim 1 wherein said nucleic acid coupled to said first reactant is selected from the group consisting of single-stranded RNA, single-stranded DNA and double-stranded DNA.

7. The method of claim 1 wherein said nucleic acid test mixture comprises pyrimidines modified at the 2'"'"'- or 5-positions.

8. The method of claim 1 wherein said nucleic acid test mixture comprises purines modified at the 8-position.

9. The method of claim 1 which further comprises a linker group coupled between said first reactant and said nucleic acid.

10. The method of claim 9 wherein said linker group has a size in the range of 10 to 1000.

11. The method of claim 10 wherein said linker group is selected from the group consisting of PEG, polyvinyl alcohol, polyacrylates and polypeptides.

12. The method of claim 1 wherein said first reactant is a dienophile, said free reactant is a diene, and said product is a cyclohexene.

2. A method for coproducing a nucleic acid that facilitates bond formation and a product that performs a preselected function on a target comprising:

(a) preparing a nucleic acid test mixture;

(b) coupling a first reactant consisting of a small organic molecule with a molecular weight in the range of 2 to 1000 to each member of said nucleic acid test mixture to form a nucleic acid-first reactant test mixture;

(c) forming a product library by contacting said nucleic acid-first reactant test mixture with a mixture of free reactants consisting of small organic molecules with a molecular weight in the range of 2 to 1000, wherein said product library is formed as a result of a bond formation reaction between said first reactant and at least one of said free reactants, wherein said bond formation is facilitated by a nucleic acid which is coupled to said first reactant and wherein each product is coupled to a nucleic acid;

(d) contacting said product library with a target, wherein products that perform a preselected function on said target relative to the product library may be partitioned from the remainder of the product library; and

(e) amplifying the nucleic acid associated with the product that performs a preselected function on said target, to yield a mixture of nucleic acids enriched for nucleic acids that facilitate bond formation between said first reactant and said free reactant, whereby said nucleic acid that facilitates bond formation and said product are produced.

3. The method of claim 2 which further comprises:

(f) coupling the amplified nucleic acids with said first reactant; and

(g) repeating steps (c) through (f) until the product having said ability to perform said preselected function on said target is produced in sufficient quantity for structure determination.

13. The method of claim 2 wherein said nucleic acid test mixture comprises nucleic acids having a region of conserved sequences and a region of randomized sequences.

14. The method of claim 2 wherein said nucleic acid coupled to said first reactant is selected from the group consisting of single-stranded RNA, single-stranded DNA and double-stranded DNA.

15. The method of claim 2 wherein said nucleic acid test mixture comprises pyrimidines at the 2'"'"'- or 5- positions.

16. The method of claim 2 wherein said nucleic acid test mixture comprises purines modified at the 8-position.

17. The method of claim 2 which further comprises a linker group coupled between said first reactant and said nucleic acid.

18. The method of claim 17 wherein said linker group has a size in the range of 10 to 1000.

19. The method of claim 18 wherein said linker group is selected from the group consisting of PEG, polyvinyl alcohol, polyacrylates and polypeptides.

20. The method of claim 2 wherein said first reactant is a dienophile, said free reactant is a diene, and said product is a cyclohexene.

4. A method for producing a product library comprising contacting a mixture of first reactants each coupled to a member of a nucleic acid test mixture of free reactants, each first and free reactant consisting of a small organic molecule with a molecular weight in the range of 2 to 1000, wherein said product library is formed as a result of a bond formation reaction between said first reactant and at least one of said free reactants, wherein said bond formation reaction is facilitated by the nucleic acid coupled to said first reactant.