Truncation SELEX method

• US 6,855,496 B2
• Filed: 07/17/2001
• Issued: 02/15/2005
• 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): Forman, B J
• Application Number:
  US09/907,111
• Publication Number:
  US 20030003461A1
• Time in Patent Office: 1,309 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3
• US Class Current: 435/5
• 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 of identifying a single stranded RNA ligand of a target molecule from a candidate mixture of single-stranded RNA molecules each comprising a region of randomized sequence, said method comprising:

a) contacting said candidate mixture with said target molecule;

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

c) adding a first tail sequence to said RNA molecules partitioned in step b) using terminal transferase;

d) reverse transcribing said tailed RNA molecules to cDNA using a primer complementary to said first tail sequence;

e) adding a second tail sequence to said cDNA molecule using terminal transferase, wherein said first and said second tail sequences have different sequences;

f) amplifying said tailed cDNA molecules of e) using said primer of step d) and a primer comprising sequence complementary to said second tail sequence and further comprising an RNA polymerase promoter sequence, whereby double stranded DNA template is produced;

g) transcribing said double stranded DNA template off) to yield single-stranded RNA molecules; and

h) removing said tail sequences from said single-stranded RNA molecules of g) whereby a single-stranded RNA ligand of said target molecule is identified.

2. The method of claim 1 further comprising repeating steps a) through h).

3. A method of identifying a single stranded RNA ligand of a target molecule from a candidate mixture of single-stranded RNA molecules each comprising a region of randomized sequence, said method comprising:

a) contacting said candidate mixture with said target molecule;

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

c) attaching a first oligonucleotide to the 3′

end of said RNA molecules partitioned in step b);

d) reverse transcribing said RNA molecules of c) to form cDNA using a primer complementary to said first oligonucleotide;

e) attaching a double-stranded deoxy oligonucleotide to said cDNA, said double-stranded deoxy oligonucleotide comprising a RNA polymerase promoter;

f) amplifying the cDNA of step e) to form a double-stranded DNA template;

g) transcribing said amplified double-stranded DNA template to yield single-stranded RNA molecules; and

h) removing said first oligonucleotide sequence from said single-stranded RNA molecules of g) whereby a single-stranded RNA ligand of said target molecule is identified.

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

5. 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 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;

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

d) optionally repeating steps a)-c) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of ligand enrichment;

e) randomly fragmenting the amplified increased affinity nucleic acids;

f) inserting fragments of a predetermined length into a circular vector; and

g) amplifying the inserted fragments using primers complementary to sequences in said circular vector that flank said inserted fragments, whereby nucleic acid ligands of said target having said predetermined length are identified.

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

7. The method of claim 5 wherein said candidate mixture is comprised of DNA.

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

9. The method of claim 7 wherein said deoxyribonucleic acids are modified deoxyribonucleic acids.