Truncation selex method

• US 6,261,774 B1
• Filed: 03/24/1999
• Issued: 07/17/2001
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: Gilead Sciences Inc.
• Inventors: Javornik, Brenda, Shtatland, Timur, Gold, Larry, Pagratis, Nikos
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US09/275,850
• Time in Patent Office: 846 Days
• Field of Search: 435/6, 435/91.2, 536/25.4
• 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/155 : incorporating/generating a new priming site
• 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 derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin G01N2333/665, corticotropin G01N2333/695
• 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 is directed to a method for identifying nucleic acid ligands by the SELEX method wherein the participation of fixed sequences is eliminated or minimized.

Claims

1. A method for identifying nucleic acid ligands of a target molecule from a candidate mixture comprised of single stranded nucleic acids each having a region of randomized sequence and a region of fixed sequence, said method comprising:

a) preparing a candidate mixture of single-stranded nucleic acids wherein each nucleic acid member of said candidate mixture comprises a region of randomized sequence and a region of fixed sequence;

b) annealing oligonucleotides to the fixed sequences that are complementary to said fixed sequences to form duplex regions, wherein said fixed sequences of said duplex regions are unavailable for binding to said target;

c) contacting said candidate mixture with said target molecule;

d) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and

e) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids whereby nucleic acid ligands of the target molecule are identified.

2. A method for identifying nucleic acid ligands of a target molecule from a candidate mixture comprised of single stranded nucleic acids each having a region of randomized sequence and one or more regions of fixed sequences, said method comprising:

a) preparing a candidate mixture of single-stranded nucleic acids wherein each nucleic acid member of said candidate mixture comprises a region of randomized sequence and one or more regions of fixed sequences;

b) contacting said candidate mixture with said target molecule;

c) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture;

d) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids;

e) replacing said one or more regions of fixed sequences with a different one or more regions of fixed sequences, whereby the participation of any given fixed sequence in binding to the target is minimized; and

f) repeating steps b)-d), whereby nucleic acid ligands of the target molecule are identified.

3. A method for identifying nucleic acid ligands of a target molecule from a candidate mixture comprised of single stranded nucleic acids each having a region of randomized sequence, said method comprising:

a) contacting said candidate mixture with said target molecule;

b) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture;

c) hybridizing the nucleic acids partitioned in step b) with a library of single stranded nucleic acids that are complementary to the single stranded nucleic acids of the candidate mixture, wherein each nucleic acid member of the complementary library has a fixed region, and wherein the fixed region facilitates amplification of the nucleic acids partitioned in step b);

d) amplifying the nucleic acids that hybridized to a nucleic acid in the complementary library whereby increased affinity nucleic acid ligands of the target molecule are produced, wherein the increased affinity nucleic acid ligands further comprise said fixed region that facilitates amplification; and

e) cleaving said increased affinity nucleic acids to remove said fixed region that facilitates amplification whereby nucleic acid ligands of the target molecule are identified.

4. The method of claim 3 further comprising repeating steps a) through e).

5. The method of claim 3 wherein said candidate mixture is comprised of RNA.

6. The method of claim 3 wherein said candidate mixture is comprised of DNA.

7. The method of claim 5 wherein said RNA acids are modified ribonucleic acids.

8. The method of claim 6 wherein said DNA acids are modified ribonucleic acids.

9. The method of claim 3 wherein the fixed regions of the increased affinity nucleic acids are cleaved by a restriction enzyme.

10. The method of claim 9 wherein the restriction enzyme is RNase H.