High affinity nucleic acid ligands of ICP4

• US 5,795,721 A
• Filed: 01/25/1996
• Issued: 08/18/1998
• Est. Priority Date: 06/11/1990
• Status: Expired due to Fees

• Current Assignee: Gilead Sciences Inc.
• Original Assignee: NeXstar Pharmaceuticals, Inc. (Gilead Sciences Inc.)
• Inventors: Rabin, Ross S., Gold, Larry, Jayasena, Sumedha D.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/591,989
• Time in Patent Office: 936 Days
• Field of Search: 435/91.2, 435/6, 435/91.1, 536/23.1, 436/501
• 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 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 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

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to Transcripion Regulatory Factors, specifically ICP4. Included in the invention are specific ssDNA and RNA ligands to ICP4 identified by the SELEX method.

Claims

1. A method of identifying nucleic acid ligands to an ICP4 protein family member, comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture of nucleic acids with an ICP4 protein family member, wherein nucleic acids having an increased affinity to an ICP4 protein family member relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding an ICP4 protein family member, wherein nucleic acid ligands of an ICP4 protein family member may be identified.

2. The method of claim 1 further comprising:

e) repeating steps b), c), and d).

3. The method of claim 1 wherein the ICP4 protein family member is ICP4.

4. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

5. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded ribonucleic acids.

6. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded deoxyribonucleic acids.

7. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of double stranded nucleic acids.

8. A purified and isolated non-naturally occurring nucleic acid ligand to a Transcription Regulatory Factor identified according to the method of claim 1.

10. The nucleic acid ligand to ICP4 of claim 10 identified according to the method of claim 1.

11. The nucleic acid ligand to ICP4 of claim 10 identified according to the method of claim 2.

12. The nucleic acid ligand of claim 10, wherein said ligand is ssDNA selected from the group consisting of the sequences set forth in Tables 5 and 7-8 (SEQ ID NOS:

17-57, 59-74).

13. The nucleic acid ligand of claim 10, wherein said ligand is an RNA selected from the group consisting of the sequences set forth in Table 9 (SEQ ID NOS:

75-86).

9. A purified and isolated non-naturally occurring nucleic acid ligand to ICP4.