Riboregulator compositions and methods of use

Patent metadata

Field	Value
Publication number	US-9550987-B2
Application number	US-201314440917-A
Country	US
Kind code	B2
Filing date	Nov 6, 2013
Priority date	Nov 6, 2012
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

The invention provides novel and versatile classes of riboregulators, including inter alia activating and repressing riboregulators, switches, and trigger and sink RNA, and methods of their use for detecting RNAs in a sample such as a well and in modulating protein synthesis and expression.

First claim

What is claimed is:

1. A toehold riboregulator comprising
an RNA comprising in a 5′ to 3′ order
(1) a single-stranded toehold domain,
(2) a fully or partially double-stranded stem domain comprising
(i) an initiation codon, and
(ii) a loop domain comprising a ribosome binding site, and

(3) a coding domain.

2. The riboregulator of claim 1 , further comprising a spacer domain.

3. The riboregulator of claim 2 , wherein the spacer domain encodes low molecular weight amino acids.

4. The riboregulator of claim 2 , wherein the spacer is about 9-33 nucleotides in length.

5. The riboregulator of claim 2 , wherein the spacer is about 21 nucleotides in length.

6. The riboregulator of claim 2 , wherein the spacer domain is situated between the stem domain and the coding domain.

7. The riboregulator of claim 1 , wherein the stem domain comprises sequence upstream (5′) and/or downstream (3′) of the initiation codon.

8. The riboregulator of claim 7 , wherein the sequence upstream of the initiation codon is about 6 nucleotides.

9. The riboregulator of claim 7 , wherein the sequence downstream of the initiation codon is about 9 nucleotides.

10. The riboregulator of claim 7 , wherein the sequence downstream of the initiation codon does not encode a stop codon.

11. The riboregulator of claim 1 , wherein the coding domain encodes a reporter protein.

12. The riboregulator of claim 11 , wherein the reporter protein is green fluorescent protein (GFP).

13. The riboregulator of claim 1 , wherein the coding domain encodes a non-reporter protein.

14. The riboregulator of claim 1 , wherein the toehold domain is complementary in sequence to a naturally occurring RNA.

15. The riboregulator of claim 1 , wherein the toehold domain is complementary in sequence to a non-naturally occurring RNA.

16. A trans-activating RNA (taRNA) comprising
a first domain that hybridizes to a toehold domain of a riboregulator of claim 1 and that comprises no or minimal secondary structure, and
a second domain that hybridizes to a sequence in the stem domain of the riboregulator of claim 1 .

17. The trans-activating RNA of claim 16 , wherein the first domain is 100% complementary to the toehold domain.

18. A system comprising
the riboregulator of claim 1 , and
the trans-activating RNA (taRNA) of claim 16 .

19. The system of claim 18 , wherein the system is a cell.

20. The system of claim 19 , wherein the cell is a prokaryotic cell.

21. The system of claim 18 , wherein the system is a cell-free in vitro system.

22. The system of claim 18 , wherein the riboregulator and the taRNA are hybridized to each other.

23. The system of claim 18 , wherein the ratio of riboregulator to taRNA is less than 1, less than 0.5, or less than 0.1.

24. The system of claim 18 , wherein the riboregulator is comprised in a first nucleic acid and the taRNA is comprised in a second nucleic acid.

25. The system of claim 24 , wherein the first nucleic acid is a first plasmid and the second nucleic acid is a second plasmid.

26. The system of claim 24 , wherein the first plasmid comprises a medium copy origin of replication and the second plasmid comprises a high copy origin of replication.

27. A nucleic acid comprising
the riboregulator of claim 1 .

28. A host cell comprising the nucleic acid of claim 27 .

29. A nucleic acid comprising
the trans-activating RNA (taRNA) of claim 16 .

30. A host cell comprising the nucleic acid of claim 29 .

31. A method of detecting presence of an RNA in a sample, comprising
combining a riboregulator of claim 1 with a sample, wherein the riboregulator comprises a toehold domain that is complementary to an endogenous RNA, and wherein the riboregulator comprises a coding domain that encodes a reporter protein, under conditions that allow translation of the coding domain in the presence of the endogenous RNA but not in the absence of the endogenous RNA, and
detecting the reporter protein as an indicator of the endogenous RNA.

32. A method of detecting presence of an RNA in a cell, comprising
introducing into the cell a riboregulator of claim 1 , wherein the riboregulator comprises a toehold domain that is complementary to an endogenous RNA in the cell, and wherein the riboregulator comprises a coding domain that encodes a reporter protein, and
detecting the reporter protein as an indicator of the endogenous RNA.

33. The method of claim 31 , wherein the reporter protein is green fluorescent protein (GFP).

34. The method of claim 31 , wherein amount of reporter protein is an indicator of amount of endogenous RNA.

35. A method of controlling protein translation, comprising
combining a riboregulator of claim 1 with a taRNA of claim 16 , wherein the riboregulator comprises a toehold domain that is complementary to the taRNA, and wherein the riboregulator comprises a coding domain that encodes a non-reporter protein, under conditions that allow translation of the coding domain in the presence of the taRNA but not in the absence of the taRNA.

Assignees

• Harvard College
• Univ Boston

Inventors

• Green Alexander A
• Yin Peng
• Collins James J

Classifications

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12Q1/6816

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title

• C12N2320/12

  in functional genomics, i.e. for the determination of gene function · CPC title

• C12N15/11 Primary

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12Q1/6897

  involving reporter genes operably linked to promoters · CPC title