Target-dependent transcription using deletion mutants of N4 RNA polymerase

• US 20040191812A1
• Filed: 12/23/2003
• Published: 09/30/2004
• Est. Priority Date: 05/22/2001
• Status: Active Grant

• Current Assignee: Epicenter Technologies, University of Chicago
• Original Assignee: University of Chicago
• Inventors: Dahl, Gary A., Jendrisak, Jerome J., Rothman-Denes, Lucia B., Davydova, Elena K., Gerdes, Svetlana Y.
• Granted Patent: US 7,838,270 B2
• Time in Patent Office: Days
• US Class Current: 506/10
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention comprises novel methods, compositions and kits that use N4 vRNAP deletion mutants to detect and quantify analytes comprising one or multiple target nucleic acid sequences, including target sequences that differ by as little as one nucleotide or non-nucleic acid analytes, by detecting a target sequence tag that is joined to an analyte-binding substance. The method consists of an annealing process, a DNA ligation process, an optional DNA polymerase extension process, a transcription process, and, optionally, a detection process

Claims

1. ) A method for detecting a target nucleic acid sequence, the method comprising:

a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′

-end of a first target-complementary sequence is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end of a second target-complementary sequence is complementary to the 3′

-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′

- of the first target-complementary sequence, wherein the promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter;

b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the target nucleic acid sequence to form a target probe-target complex;

c) contacting the target probe-target complex with a ligase under ligation conditions to form a transcription substrate;

d) contacting the transcription substrate with the RNA polymerase;

e) optionally, repeating steps (a) through (e); and

f) detecting the transcription product.

2. ) The method of claim 1, wherein the target nucleic acid sequence comprises a single-stranded DNA molecule obtained by reverse transcription of RNA.

3. ) The method of claim 1, wherein the target nucleic acid sequence comprises a DNA target nucleic acid in a sample.

4. ) The method of claim 1, wherein the method is used for detecting an analyte in a sample, wherein the target nucleic acid sequence comprises a target sequence tag that is joined to an analyte-binding substance, the method further comprising prior to step (a) contacting the analyte-binding substance with the analyte to form a specific binding pair and separating the specific binding pair from analyte-binding substance molecules that are not bound to the analyte.

5. ) The method of claim 4, wherein the analyte is selected from the group consisting of a biochemical molecule, a biopolymer, a protein, a glycoprotein, a lipoprotein, an enzyme, a hormone, a biochemical metabolite, a receptor, an antigen, an antibody, a nucleic acid, a DNA molecule, an RNA molecule, a polysaccharide and a lipid.

6. ) The method of claim 4, wherein the analyte-binding substance is selected from the group consisting of a nucleic acid, a polynucleotide, an oligonucleotide, a segment of a nucleic acid or polynucleotide, a DNA molecule, an RNA molecule, a molecule comprising both DNA and RNA mononucleotides, modified DNA mononucleotides, a molecule obtained by a method termed “

SELEX”

, a nucleic acid molecule having an affinity for protein molecules, a polynucleotide molecule having an affinity for protein molecules, an operator, a promoter, an origin of replication, a ribosomal nucleic acid sequence, a sequence recognized by steroid hormone-receptor complexes, a peptide nucleic acid (PNA), a nucleic acid and a PNA, a molecule prepared by using a combinatorial library of randomized peptide nucleic acids, an oligonucleotide or polynucleotide with a modified backbone that is not an amino acid, a lectin, a receptor for a hormone, a hormone, and an enzyme inhibitor.

7. ) The method of claim 1, wherein the target nucleic acid sequence comprises a DNA target nucleic acid that is a product of an amplification reaction.

8. ) The method of claim 1 wherein the target nucleic acid sequence comprises a product of rolling circle replication.

9. ) The method of claim 7, wherein the amplification reaction is selected from the group consisting of PCR, RT-PCR, NASBA, TMA, 3SR, LCR, LLA, SDA, Multiple Displacement Amplification, ICAN™

, UCAN™

, Loop-AMP, SPIA™ and

Ribo-SPIA™

.

10. ) The method of claim 1, wherein the one or more target probes comprise a bipartite target probe.

11. ) The method of claim 1 wherein the target probe comprising the second target-complementary sequence also comprises a signal sequence 5′

- of said target-complementary sequence.

12. ) The method of claim 11 wherein the signal sequence comprises a substrate for Q-beta replicase.

13. ) The method of claim 11 wherein the signal sequence comprises a sequence that encodes a detectable protein.

14. ) The method of claim 13 wherein the detectable protein is green fluorescent protein.

15. ) The method of claim 11 wherein the signal sequence comprises a sequence that is detectable by a probe.

16. ) The method of claim 11 wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.

17. ) The method of claim 10 wherein the bipartite target probe comprises a transcription termination sequence 5′

- of the second target-complementary sequence.

18. ) The method of claim 10 wherein the bipartite target probe comprises two target-complementary sequences that can anneal adjacently to the target nucleic acid sequence.

19. ) The method of claim 1, wherein the one or more target probes comprise a promoter target probe comprising the first target-complementary sequence and a signal target probe comprising the second target-complementary sequence.

20. ) The method of claim 19 wherein the signal target probe comprises a signal sequence 5′

- of the second target-complementary sequence.

21. ) The method of claim 20 wherein the signal sequence comprises a substrate for Q-beta replicase.

22. ) The method of claim 20 wherein the signal sequence comprises a sequence that encodes a detectable protein.

23. ) The method of claim 22 wherein the detectable protein is green fluorescent protein.

24. ) The method of claim 20 wherein the signal sequence comprises a sequence that is detectable by a probe.

25. ) The method of claim 20 wherein the signal sequence comprises a sequence that is detectable by a molecular beacon.

26. ) The method of claim 19 wherein the first target-complementary sequence and the second target-complementary sequence can anneal adjacently to the target nucleic acid sequence.

27. ) The method of claim 1 wherein one target probe is provided.

28. ) The method of claim 1 wherein two target probes are provided.

29. ) The method of claim 1 wherein the number of provided target probes is selected from the group consisting of 3, 4, 5, 6, 7, 8, 9, and 10.

30. ) The method of claim 1 wherein the promoter is an N4 vRNAP promoter set forth in SEQ ID NO:

16, SEQ ID NO;

19, SEQ ID NO;

27, SEQ ID NO;

28 or SEQ ID NO;

29.

31. ) The method of claim 1 wherein the promoter is a P2 sequence set forth in SEQ ID NO:

16 or SEQ ID NO;

28.

32. ) The method of claim 1 wherein the ligase is Ampligase®

Thermostable DNA Ligase.

33. ) The method of claim 1 wherein the ligase is selected from the group consisting of Tfl DNA Ligase, Tsc DNA Ligase, Pfu DNA ligase, T4 DNA ligase and Tth DNA ligase.

34. ) The method of claim 1 wherein the RNA polymerase comprises a region encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO:

2, SEQ ID NO;

4, SEQ ID NO;

6, SEQ ID NO;

8 or SEQ ID NO;

15.

35. ) The method of claim 1 wherein the RNA polymerase comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

5, SEQ ID NO;

7 or SEQ ID NO;

14.

36. ) The method of claim 1 wherein the RNA polymerase comprises an N4 min-vRNAP.

37. ) The method of claim 1 wherein the RNA polymerase comprises a domain having 1,106 amino acid residues corresponding to amino acid residues 998-2103 of N4 vRNAP.

38. ) The method of claim 1 wherein the transcription product comprises only ribonucleotides.

39. ) The method of claim 1 wherein the transcription product comprises at least one pyrimidine 2′

-deoxyribonucleotide having a 2′

-substituent on the sugar moiety.

40. ) The method of claim 1 wherein the transcription product comprises at least one pyrimidine 2′

-fluoro-2′

-deoxyribonucleotide.

41. ) The method of claim 1 wherein the transcription product comprises pyrimidine 2′

-fluoro-2′

-deoxyribonucleotides.

42. ) The method of claim 1 wherein the transcription product comprises AMP, GMP, 2′

-F-dUMP and 2′

-F-dCMP.

43. ) The method of claim 1 wherein the transcription product comprises at least one 2′

-amino-2′

-deoxyribonucleotide.

44. ) The method of claim 1 wherein the transcription product comprises at least one 2′

-methoxy-2′

-deoxyribonucleotide.

45. ) The method of claim 1 wherein the transcription product comprises at least one 2′

-azido-2′

-deoxyribonucleotide.

46. ) The method of claim 1 wherein the transcription product comprises at least one 2′

-amino-2′

-deoxyribonucleotide.

47. ) A method for detecting a target nucleic acid sequence, the method comprising:

a) providing a target sequence amplification probe (TSA probe) comprising a linear single-stranded DNA molecule comprising a 5′

-end portion and a 3′

-end portion that are not joined, wherein the 5′

-end portion is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end portion is complementary to the 3′

-end of the target nucleic acid sequence;

b) providing a primer that is complementary to the TSA probe;

c) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′

-end of a first target-complementary sequence is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end of a second target-complementary sequence is complementary to the 3′

-end of the target nucleic acid sequence; and

wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′

- of the first target-complementary sequence, wherein the promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter;

d) contacting the TSA probe with the target nucleic acid sequence and incubating under hybridization conditions, such that the end portions anneal adjacently to the target nucleic acid sequence to form a TSA probe-target complex;

e) contacting the TSA probe-target complex with a ligase under ligation conditions, such that a target sequence amplification circle (TSA circle) is formed;

f) contacting the TSA circle with the primer and incubating under hybridization conditions to form a TSA circle-primer complex;

g) contacting the TSA circle-primer complex with a strand-displacing DNA polymerase under strand-displacing polymerization conditions, such that a rolling circle replication product comprising multiple copies of the target nucleic acid sequence is formed;

h) contacting the target probes with the rolling circle replication product and incubating under hybridization conditions, such that the target-complementary sequences anneal adjacently to the rolling circle replication product to form a rolling circle replication product-target complex;

i) contacting the rolling circle replication product-target complex with a ligase under ligation conditions to form a transcription substrate;

j) optionally, releasing the transcription substrate from the rolling circle replication product complex,

k) contacting the transcription substrate with the RNA polymerase under transcription conditions to form a transcription product;

l) optionally, repeating steps (a) through (k); and

m) detecting the transcription product.

48. ) A method for selectively transcribing a target nucleic acid sequence, the method comprising a DNA ligation operation and a transcription operation, wherein the DNA ligation operation comprises ligation of one or more target probes comprising a promoter that is 3′

- of a target complementary sequence, which promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter to form a transcription substrate, wherein the ligation is dependent on hybridization of the target probes to the target nucleic acid sequence, to form a transcription substrate and wherein the transcription operation comprises contacting the transcription substrate with the RNA polymerase.

49. ) A method for detecting a target nucleic acid sequence, the method comprising:

a) providing one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′

-end of a first target-complementary sequence is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end of a second target-complementary sequence is complementary to the 3′

-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′

-end of the first target-complementary sequence, which promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter;

b) contacting the target probes with the target nucleic acid sequence and incubating under hybridization conditions, such that the target probes anneal to the target nucleic acid sequence to form a target probe-target complex;

c) contacting the target probe-target complex with a DNA polymerase under DNA polymerization conditions to form one or more DNA polymerase extension products that are adjacent to the 5′

-end of a target-probe, such that a complex is formed;

d) contacting the complex with a ligase under ligation conditions to form a transcription substrate;

e) contacting the transcription substrate with the RNA polymerase to form a transcription product;

f) optionally, repeating steps (a) through (f); and

g) detecting the transcription product.

50. ) A kit for detecting a target nucleic acid sequence, the kit comprising:

a) one or more target probes comprising a linear single-stranded DNA molecule, the target probes comprising at least two target-complementary sequences that are not joined to each other, wherein the 5′

-end of a first target-complementary sequence is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end of a second target-complementary sequence is complementary to the 3′

-end of the target nucleic acid sequence, and wherein the target probe that comprises the first target-complementary sequence also comprises a promoter that is joined to the 3′

- of the first target-complementary sequence, wherein the promoter binds an RNA polymerase that lacks helicase-like activity and that can transcribe RNA using a single-stranded promoter;

b) a ligase; and

c) the RNA polymerase.

51. ) The kit of claim 50 further comprising a reverse transcriptase.

52. ) The kit of claim 50 further comprising a target sequence amplification probe (TSA probe) comprising a linear single-stranded DNA molecule comprising a 5′

-end portion and a 3′

-end portion that are not joined, wherein the 5′

-end portion is complementary to the 5′

-end of the target nucleic acid sequence, and wherein the 3′

-end portion is complementary to the 3′

-end of the target nucleic acid sequence;

a primer that is complementary to the target sequence amplification probe, and a strand-displacing DNA polymerase.

53. ) The kit of claim 50 further comprising a DNA polymerase.