Assays for measuring nucleic acid binding proteins and enzyme activities

• US 6,312,896 B1
• Filed: 09/17/1998
• Issued: 11/06/2001
• Est. Priority Date: 09/17/1998
• Status: Expired due to Fees

• Current Assignee: BioVeris Corporation (Roche Holding AG)
• Original Assignee: IGEN International, Inc. (Roche Holding AG)
• Inventors: Kibbey, Maura C., Kenten, John H., Heroux, Jeffrey A.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/157,808
• Time in Patent Office: 1,146 Days
• Field of Search: 435/6, 435/7.1, 435/7.5, 435/18, 530/350, 536/23.1
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins

Abstract

The present invention provides processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities using labeled nucleic acids of labeled protein/peptide molecules.

Claims

1. A method for assaying a sample for the presence of a nucleic acid binding protein, which comprises:

a) mixing at least one predetermined single- or double-stranded nucleic acid containing at least one label and containing a protein binding nucleotide sequence with a sample which may contain a nucleic acid binding protein,

b) incubating the mixture of step a) under conditions which allow the binding of said nucleic acid binding protein to said at least one predetermined single- or double-stranded nucleic acid to form a complex,

c) adding a nucleic acid-cleaving enzyme or reagent to the mixture of step b),

d) incubating the mixture of step c) under conditions which allow the cleavage of said at least one predetermined single- or double-stranded nucleic acid which has not formed a complex, and

e) measuring the amount of said complex by means that do not include gel electrophoretic separation to measure said nucleic acid binding protein.

2. A method as in claim 1 or 2 wherein said at least one predetermined single- or double-stranded nucleic acid is 4 to 1000 nucleotides in length.

4. A method as in claim 1 wherein said label is an electrochemiluminescent label.

5. A method as in claim 4 wherein said electrochemiluminescent label is RuBpy.

6. A method as in claim 1 or 2 wherein said label is selected from the group consisting of a radioactive moiety, fluorescent moiety, enzyme, chemiluminescent moiety, electrochemiluminescent moiety, bioluminescent moiety, and an optically observable particle.

7. A method as in claim 1 wherein said at least one predetermined single- or double-stranded nucleic acid sequence has at least one capture moiety attached thereto.

8. A method as in claim 7 wherein said label is RuBpy and said capture moiety is selected from the group consisting of biotin, avidin, streptavidin, antibody, antigen, lectin, receptor, ligand, hormone, nucleic acid sequence, mimitope, and a nucleic acid base pairing polymer.

9. A method as in claim 1 or 2 wherein prior to step a), at least one predetermined single- or double-stranded nucleic acid sequence is contacted with a solid phase.

10. A method as in claim 1 or 2 wherein after step d), the mixture is contacted with a solid phase.

11. A method as in claim 1 or 2 wherein said at least one predetermined single- or double-stranded nucleic acid sequence is RNA.

3. A method for assaying a sample for the presence of an inhibitor of a predetermined nucleic acid binding protein, which comprises:

a) mixing at least one predetermined single- or double-stranded nucleic acid containing at least one label and containing a protein binding nucleotide sequence and a predetermined nucleic acid binding protein with a sample which may contain an inhibitor of the binding of said predetermined nucleic acid binding protein with said at least one predetermined single- or double-stranded nucleic acid,

b) incubating the mixture of step a) under conditions which allow the binding of said nucleic acid binding protein to said at least one predetermined single- or double-stranded nucleic acid to form a complex,

c) adding a nucleic acid-cleaving enzyme or reagent to the mixture of step b), consisting of peptide nucleic acid linkages, phosphorothioate linkages, and methyl phosphonate linkages.

12. A kit comprising in one or more containers:

a) a nucleic acid having a predetermined protein binding region wherein said nucleic acid has a detectable moiety attached thereto, and wherein said nucleic acid has a plurality of nucleic acid linkages, wherein said linkages prevent cleavage of the nucleic acid by a nuclease when a protein is bound to said protein binding region,

b) a nucleic acid-cleaving enzyme or nucleic acid-cleaving reagent, and

c) a solid phase.

13. A kit as in claim 12 wherein said nucleic acid has a capture moiety attached trereto.

14. A kit as in claim 12 wherein said detectable moiety is an ECL label.

15. A kit as in claim 14 wherein said ECL label is Ru(byp).

16. A kit according to claim 12 wherein said nucleic acid linkages are selected from the group consisting of peptide nucleic acid linkages, phosphorothioate linkages and methyl phosphonate linkages.

17. A nucleic acid comprising a predetermined protein binding region wherein said nucleic acid has a detectable moiety attached thereto and wherein said nucleic acid has a plurality of nucleic acid linkages, wherein said linkages prevent cleavage of the nucleic acid by a nuclease when a protein is bound to said protein binding region.

18. A nucleic acid as in claim 17 wherein said nucleic acid has a capture moiety attached.

19. A nucleic acid of claim 17 wherein every nucleic acid linkage contained outside said protein binding region is selected from the group consisting of peptide nucleic acid linkages, phosphorothioate linkages, and methyl phosphonate linkages.

20. A nucleic acid according to claim 17 wherein said plurality of nucleic acid linkages are selected from the group consisting of peptide nucleic acid linkages, phosphorothioate linkages, and methyl phosphonate linkages.

21. A method for assaying a sample for the presence of a nucleic acid binding protein, which comprises:

a) mixing at least one predetermined single- or double-stranded nucleic acid containing modified nucleotides that are resistant to nuclease cleavage, at least one label, and containing a protein binding nucleotide sequence with a sample which may contain a nucleic acid binding protein,

b) incubating the mixture of step a) under conditions which allow the binding of said nucleic acid binding protein to said at least one predetermined single- or double-stranded nucleic acid to form a complex,

c) adding a nucleic acid-cleaving enzyme or reagent to the mixture of step b),

d) incubating the mixture of step c) under conditions which allow the cleavage of said at least one predetermined single- or double-stranded nucleic acid which has not formed a complex, and

e) measuring the amount of said complex by means that do not include gel electrophoretic separation to measure said nucleic acid binding protein.

22. A method as in claim 21 wherein said at least one predetermined single- or double-stranded nucleic acid contains from 1 to 999 modified nucleotides.

23. A method as in claim 22 wherein said modified nucleotides are not contained within said protein binding nucleotide sequence.