Methods of detecting transcription

Patent metadata

Field	Value
Publication number	US-9260703-B2
Application number	US-92061806-A
Country	US
Kind code	B2
Filing date	May 17, 2006
Priority date	May 18, 2005
Publication date	Feb 16, 2016
Grant date	Feb 16, 2016

Abstract

The present invention includes compositions, methods and kits for the real-time detection of transcription and translation in live cells, tissues and organisms. The present invention further provides method for the rapid sequencing of nucleic acids without using conventional sequencing techniques or reactions.

First claim

We claim:

1. A method of detecting transcription of a DNA molecule, the method comprising contacting a cell with a detector molecule and a chimeric RNA polymerase molecule comprising a detector binding domain that specifically binds to the detector molecule, wherein the detector molecule and the chimeric RNA polymerase molecule localize to the nucleus of the cell;
wherein the detector molecule comprises;
a) a chimeric RNA polymerase binding domain (CRPBD) capable of binding to the detector binding domain,
b) a peptide nucleic acid (PNA) complementary to a portion of an RNA molecule; and
c) a signaling moiety,
and wherein when the chimeric RNA polymerase transcribes the DNA to produce a nascent RNA molecule, the PNA binds to the nascent RNA molecule and a signal is emitted by the detector molecule, and
detecting the signal, thereby detecting transcription of the DNA molecule.

2. The method of claim 1 , wherein the detector molecule further comprises a cell penetrating peptide.

3. The method of claim 1 , wherein the detector binding domain is selected from the group consisting of an SH3 domain and a leucine zipper.

4. The method of claim 1 , wherein the CRPBD is selected from the group consisting of an α-PAK domain and a leucine zipper.

5. The method of claim 2 , wherein the cell penetrating peptide is selected from the group consisting of a transportan peptide (TP), a TP10 peptide, a pVEC peptide, a penetratin peptide, a tat fragment peptide, a signal sequence based peptide, and an amphiphilic model peptide.

6. The method of claim 1 , wherein the signaling moiety comprises a fluorescent molecule.

7. The method of claim 6 , wherein the fluorescent molecule is selected from the group consisting of ReAsH, bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine (BSR), Cy3B, Cy5, tetramethylrhodamine and fluorescein.

8. The method of claim 1 , wherein the PNA is complementary to a di-nucleotide or a tri-nucleotide of the nascent RNA molecule.

9. The method of claim 8 , wherein the PNA is complementary to a tri-nucleotide of the nascent RNA molecule.

10. The method of claim 9 , wherein the tri-nucleotide has the nucleic acid sequence CAC.

11. The method of claim 1 , wherein the chimeric RNA polymerase is a chimeric RNA polymerase II.

12. The method of claim 1 , wherein the chimeric RNA polymerase is a chimeric T7 RNA polymerase.

13. The method of claim 1 , wherein the signal is a fluorescent resonance energy transfer (FRET) signal or a polarity change signal.

14. The method of claim 1 , wherein the cell is an eukaryotic cell.

15. The method of claim 1 , wherein the cell is in an animal.

16. The method of claim 15 , wherein the animal is a mammal.

17. A method of detecting the transcription of a nucleic acid molecule, the method comprising
contacting a cell with a detector molecule and a chimeric enzyme that transcribes a nucleic acid molecule, wherein the chimeric enzyme comprises a detector binding domain that specifically binds the detector molecule, wherein the detector molecule and the chimeric enzyme localize to the nucleus of the cell, wherein the detector molecule comprises:
a) a chimeric enzyme binding domain (CEBD) capable of binding to the detector binding domain,
b) a PNA complementary to a portion of a nucleic acid molecule; and
c) a signaling moiety,
and wherein when the chimeric enzyme transcribes a nucleic acid, the PNA binds to a nascent nucleic acid molecule emerging from the enzyme and a signal is emitted by the detector molecule, and
detecting the signal, thereby detecting transcription of a nucleic acid molecule.

18. The method of claim 17 , wherein the detector molecule further comprises a cell penetrating peptide.

19. The method of claim 17 , wherein the detector binding domain is selected from the group consisting of an SH3 domain and a leucine zipper.

20. The method of claim 17 , wherein the CEBD is selected from the group consisting of an α-PAK domain and a leucine zipper.

21. The method of claim 18 , wherein the cell penetrating peptide is selected from the group consisting of a transportan peptide (TP), a TP10 peptide, a pVEC peptide, a penetratin peptide, a tat fragment peptide, a signal sequence based peptide, and an amphiphilic model peptide.

22. The method of claim 17 , wherein the signaling moiety comprises a fluorescent molecule.

23. The method of claim 22 , wherein the fluorescent molecule is selected from the group consisting of ReAsH, bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine (BSR), Cy3B, Cy5, and fluorescein.

24. The method of claim 17 , wherein the PNA is complementary to a di-nucleotide or a tri-nucleotide portion of a nucleic acid molecule.

25. The method of claim 24 , wherein the PNA is complementary to a tri-nucleotide portion of a nucleic acid molecule.

26. The method of claim 25 , wherein the tri-nucleotide has the nucleic acid sequence CAC.

27. The method of claim 17 , wherein the chimeric enzyme that transcribes a nucleic acid molecule is a chimeric RNA polymerase II.

28. The method of claim 17 , wherein the chimeric enzyme that transcribes a nucleic acid molecule is a chimeric T7 RNA polymerase.

29. The method of claim 17 , wherein the signal is fluorescent resonance energy transfer (FRET) signal or a polarity change signal.

30. The method of claim 17 , wherein the cell is an eukaryotic cell.

31. The method of claim 17 , wherein the cell is in an animal.

32. The method of claim 31 , wherein the animal is a mammal.

33. A method of identifying a nucleic acid that is transcribed, the method comprising
contacting a cell with a detector molecule and a chimeric enzyme that transcribes a nucleic acid molecule, wherein the chimeric enzyme comprises a detector binding domain that specifically binds the detector molecule, wherein the detector molecule and the chimeric enzyme localize to the nucleus of the cell, wherein the detector molecule comprises:
a) a chimeric enzyme binding domain (CEBD) capable of binding to the detector binding domain,
b) a PNA complementary to a portion of the nucleic acid molecule; and
c) a signaling moiety,
and wherein when the chimeric enzyme transcribes the nucleic acid, the PNA binds to a nascent nucleic acid molecule emerging from the enzyme and a signal is emitted by the detector molecule,
detecting the signal, and
identifying the nucleic acid molecule transcribed using a constrained local dynamic time warp algorithm to match the signal detected to a library of signals associated with specific sequences, thereby identifying the nucleic acid that is transcribed.

Assignees

• Eberwine James H
• Langel Ulo
• Eiriksdottir Emelia
• Peritz Tina
• Sul Jai-Yoon
• Haydon Philip G
• Kim Junhyong
• Univ Pennsylvania

Inventors

• Eberwine James H
• Langel Ulo
• Eiriksdottir Emelia
• Peritz Tina
• Sul Jai-Yoon
• Haydon Philip G
• Kim Junhyong

Classifications

• C12Y207/07006

  DNA-directed RNA polymerase (2.7.7.6) · CPC title

• C12N9/96 Primary

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12N9/127 Primary

  RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase · CPC title

• C07K2319/72

  containing SH2 domain · CPC title

• C12Q1/68

  involving nucleic acids · CPC title