Nucleic acid ligands to HIV-RT and HIV-1 rev

• US 5,496,938 A
• Filed: 10/21/1992
• Issued: 03/05/1996
• Est. Priority Date: 06/11/1990
• Status: Expired due to Term

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Gold, Larry, Tuerk, Craig
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/964,624
• Time in Patent Office: 1,231 Days
• Field of Search: 435/6, 536/22.1
• US Class Current: 536/22.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 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: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/72 for hormones for neuromediators C07K14/70571
• CPC Class Codes: C07K 7/22 Tachykinins, e.g. Eledoisins , Substance P; Related peptides
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• 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 2740/16222 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• 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 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2525/101 : incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/301 : Hairpin oligonucleotides
• 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 2333/918 : Carboxylic ester hydrolases (3.1.1)
• 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

Methods for the identification and production of improved nucleic acid ligands are based on the SELEX process. Nucleic acid ligands to HIV-RT and HIV-1 Rev are identified according to the methods described herein.

Claims

1. A non-naturally occurring nucleic acid ligand to HIV reverse transcriptase (RT) obtained according to a method for identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said method comprising:

a) contacting the candidate mixture with HIV-RT, wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids 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, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of HIV-RT may be identified and obtained nucleic acids.

2. The nucleic acid ligand to HIV-RT obtained by the method of claim 1 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for a single residue substitution; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-RT may be obtained.

3. The nucleic acid ligand to HIV-RT may be obtained by the method of claim 1 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for the absence of one or more terminal residues; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-RT may be obtained.

4. A non-naturally occurring nucleic acid ligand to HIV-RT having the RNA sequence (SEQ ID NO:

12);

##STR1## wherein X=any base, and X-X'"'"' indicates a preferred base-pair.

5. A non-naturally occurring nucleic acid ligand to HIV-RT obtained according to a method for identifying an extended nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of HIV-RT comprising:

a) contacting the candidate mixture with HIV-RT, wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids 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) repeating steps a)-c), as necessary, to identify said nucleic acid ligand to HIV-RT;

e) contacting a second candidate mixture of nucleic acids with HIV-RT, wherein said second candidate mixture is comprised of nucleic acids having a fixed region and a randomized region, said fixed region corresponding to said nucleic acid ligand to HIV-RT identified in d), and wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids may be partitioned from the remainder of the candidate mixture;

f) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

g) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; and

h) repeating steps e)-h), as necessary, whereby said nucleic acid ligand to HIV-RT may be obtained.

6. A non-naturally occurring nucleic acid ligand obtained by the method of claim 5 having the RNA sequence (SEQ ID NO:

13);

##STR2## or the corresponding DNA sequence thereof;

wherein x is any base, and Z is selected from the group consisting of the sequences set forth in FIG. 9 (SEQ ID NO;

14-34).

7. A non-naturally occurring nucleic acid ligand to HIV-1 Rev obtained according to a method for identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said method comprising:

a) contacting the candidate mixture with HIV-1 Rev, wherein nucleic acids having an increased affinity to HIV-1 Rev relative to the candidate mixture nucleic acids 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, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to HIV-1 Rev may be identified and obtained.

8. The nucleic acid ligand to HIV-1 Rev obtained by the method of claim 7 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for a single residue substitution; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand.

9. The nucleic acid ligand to HIV-1 Rev obtained by the method of claim 7 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for the absence of one or more terminal residues; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-1 Rev may be obtained.

10. A non-naturally occurring nucleic acid ligand to HIV-1 Rev having a RNA sequence (SEQ ID NO:

35);

##STR3## wherein X=any base. f5 whereby nucleic acid ligands to HIV-RT may be obtained