Allosteric nucleic acid sensor molecules

• US 20040009510A1
• Filed: 04/23/2003
• Published: 01/15/2004
• Est. Priority Date: 03/06/2000
• Status: Abandoned Application

• Current Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Original Assignee: Sirna Therapeutics, Inc. (Alnylam Pharmaceuticals Incorporated)
• Inventors: Kossen, Karl, Zinnen, Shawn, Seiwert, Scott, Jadhav, Vasant, Vaish, Narendra
• Application Number:
  US10/422,050
• Publication Number:
  US 20040009510A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• 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: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/121 Hammerhead
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/317 with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/332 Abasic residue
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

Nucleic acid sensor molecules and methods are provided for the detection and amplification of signaling agents using enzymatic nucleic acid constructs, including Halfzymes, multicomponent nucleic acid sensor molecules, hammerhead enzymatic nucleic acid molecules, inozymes, G-cleaver enzymatic nucleic acid molecules, zinzymes, amberzymes and DNAzymes. Also provided are kits for detection and amplification. The nucleic acid sensor molecules, methods and kits provided herein can be used in diagnostics, nucleic acid circuits, nucleic acid computers, therapeutics, target validation, target discovery, drug optimization, single nucleotide polymorphism (SNP) detection, single nucleotide polymorphism (SNP) scoring, and proteome scoring as well as other uses described herein.

Claims

1. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and a separate effector component, wherein the enzymatic nucleic acid is assembled from two or more separate nucleic acid molecules, wherein the separate effector component is one of the two or more separate nucleic acid molecules that make up the enzymatic nucleic acid component of the nucleic acid sensor molecule, such the in the presence of the separate effector component, the enzymatic nucleic acid component assembles in a form necessary to enable the nucleic acid sensor molecule to catalyze a chemical reaction involving one or more reporter molecules, and wherein the effector and the reporter molecules are separate molecules.

2. The nucleic acid sensor molecule of claim 1, wherein the chemical reaction is a ligation reaction.

3. The nucleic acid sensor molecule of claim 2, wherein the ligation reaction involves covalent attachment of a first reporter molecule to a second reporter molecule.

4. The nucleic acid sensor molecule of claim 2, wherein the ligation reaction results in the formation of a phosphodiester bond.

5. The nucleic acid sensor molecule of claim 3, wherein the first or second reporter molecule independently comprises a terminal phosphate group.

6. The nucleic acid sensor molecule of claim 1, wherein the chemical reaction is a phosphodiester cleavage reaction.

7. The nucleic acid sensor molecule of claim 1, wherein the reporter molecule comprises one or more polynucleotides.

8. The nucleic acid sensor molecule of claim 1, wherein the enzymatic nucleic acid component is assembled from two separate nucleic acid molecules.

9. The nucleic acid sensor molecule of claim 1, wherein the enzymatic nucleic acid component is assembled from three separate nucleic acid molecules.

10. A method, comprising:

(a) contacting the nucleic acid sensor molecule of claim 1 with a system under conditions suitable for the nucleic acid sensor molecule and to catalyze a chemical reaction on a reporter molecule; and

(b) assaying for the chemical reaction on the reporter molecule.

11. The method of claim 10, wherein the chemical reaction is indicative of the presence of a target nucleic acid in the system.

12. The method of claim 10, wherein the chemical reaction is indicative of the system lacking a target nucleic acid.

13. The nucleic acid sensor molecule of claim 1, wherein the effector component is an RNA or DNA derived from a bacteria, virus, fungi, plant or mammalian genome.

14. The method of claim 11, wherein the target nucleic acid is an RNA or DNA derived from a bacteria, virus, fungi, plant or mammalian genome.

15. The nucleic acid sensor molecule of claim 1, wherein the effector component comprises a sequence derived from the Hepatitis C virus (HCV).

16. The method of claim 11, wherein the target nucleic acid comprises a sequence derived from the Hepatitis C virus (HCV) 5′

-UTR.

17. The nucleic acid sensor molecule of claim 15, wherein the Hepatitis C virus (HCV) sequence is derived from the 5′

-UTR.

18. The method of claim 16, wherein the Hepatitis C virus (HCV) sequence is derived from the 5′

-UTR.

19. A kit comprising the nucleic acid sensor molecule of claim 1.

26. A method comprising:

a. contacting the nucleic acid sensor molecule of claim 1 with a system comprising at least one ssRNA having a single nucleotide polymorphism (SNP) under conditions suitable for the enzymatic nucleic acid component of the nucleic acid sensor molecule to catalyze a chemical reaction resulting in a detectable response; and

b. assaying for said chemical reaction.

27. A method comprising:

a. contacting the nucleic acid sensor molecule of claim 2 with a system comprising at least one ssDNA having a single nucleotide polymorphism (SNP) under conditions suitable for the enzymatic nucleic acid component of the nucleic acid sensor molecule to catalyze a chemical reaction resulting in a detectable response; and

b. assaying for said chemical reaction resulting in a detectable response.

20. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded RNA (ssRNA) having a single nucleotide polymorphism (SNP) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in a detectable response.

28. The nucleic acid sensor molecule of claim 20 or claim 21, wherein said chemical reaction is cleavage of a phosphodiester internucleotide linkage.

29. The nucleic acid sensor molecule of claim 20 or claim 21, wherein said chemical reaction is ligation of a predetermined nucleic acid molecule to the nucleic acid sensor molecule.

30. The nucleic acid sensor molecule of claim 20 or claim 21, wherein said chemical reaction is ligation of a predetermined nucleic acid molecule to another predetermined nucleic acid molecule.

21. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded DNA (ssDNA) having a single nucleotide polymorphism (SNP) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in a detectable response.

22. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded RNA (ssRNA) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in cleavage of a predetermined nucleic acid molecule associated with a disease.

23. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded DNA (ssDNA) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in cleavage of a predetermined nucleic acid molecule associated with a disease.

24. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded RNA (ssRNA) having a single nucleotide polymorphism (SNP) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in ligation of a predetermined nucleic acid molecule to another predetermined nucleic acid molecule.

25. A nucleic acid sensor molecule comprising an enzymatic nucleic acid component and one or more sensor components wherein, in response to an interaction of a single stranded DNA (ssDNA) having a single nucleotide polymorphism (SNP) with the nucleic acid sensor molecule in a system, the enzymatic nucleic acid component catalyzes a chemical reaction resulting in ligation of a predetermined RNA molecule to another predetermined RNA molecule.