Cis/trans riboregulators

Patent metadata

Field	Value
Publication number	US-9534224-B2
Application number	US-53512803-A
Country	US
Kind code	B2
Filing date	Nov 14, 2003
Priority date	Nov 15, 2002
Publication date	Jan 3, 2017
Grant date	Jan 3, 2017

Abstract

The present invention provides nucleic acid molecules, DNA constructs, plasmids, and methods for post-transcriptional regulation of gene expression using RNA molecules to both repress and activate translation of an open reading frame. Repression of gene expression is achieved through the presence of a regulatory nucleic acid element (the cis-repressive RNA or crRNA) within the 5′ untranslated region (5′ UTR) of an mRNA molecule. The nucleic acid element forms a hairpin (stem/loop) structure through complementary base pairing. The hairpin blocks access to the mRNA transcript by the ribosome, thereby preventing translation. In particular, in embodiments of the invention designed to operate in prokaryotic cells, the stem of the hairpin secondary structure sequesters the ribosome binding site (RBS). In embodiments of the invention designed to operate in eukaryotic cells, the stem of the hairpin is positioned upstream of the start codon, anywhere within the 5′ UTR of an mRNA. A small RNA (trans-activating RNA, or taRNA), expressed in trans, interacts with the crRNA and alters the hairpin structure. This alteration allows the ribosome to gain access to the region of the transcript upstream of the start codon, thereby activating transcription from its previously repressed state.

First claim

We claim:

1. A recombinant prokaryotic cell comprising, a first exogenous mRNA nucleic acid sequence and a second exogenous mRNA nucleic acid sequence encoding a trans-activating RNA, wherein:
a. the first exogenous mRNA nucleic acid sequence comprises a ribosome binding site (RBS) upstream of a nucleic acid sequence encoding a gene to be expressed, and a cis-repressive nucleic acid sequence located 5′ of the RBS,
wherein the cis-repressive nucleic acid sequence comprises at least 4 nucleic acids that are complementary to, or substantially complementary to at least 4 nucleic acids in the RBS and can, upon complementary base pairing of the at least 4 nucleic acids of the cis-repressive nucleic acid sequence with the at least 4 nucleic acids in the RBS form a duplex that is part of a repressive stem-loop structure, wherein the loop of the stem-loop structure is between 4-10 nucleotides; and

b. the second exogenous nucleic acid sequence comprises an inducible promoter operatively linked to a nucleic acid sequence encoding the trans-activating RNA, wherein the trans-activating RNA comprises (i) a 5′ non-stem nucleic acid sequence, comprising at least 4 nucleotides that are complementary to, or substantially complementary to at least 4 nucleotides of the cis-responsive nucleic acid sequence, and (ii) a stem-loop structure comprising a RNA duplex and loop;

wherein:
when the trans-activating RNA is not present, the cis-repressive nucleic acid sequence and RBS form the repressive stem-loop structure, wherein the repressive stem-loop structure blocks access of a ribosome to the RBS thereby repressing the translation of the gene, and
when the trans-activating RNA is present, the 5′ non-stem nucleic acid sequence of the trans-activating RNA forms a duplex with at least 4 nucleotides of the cis-repressive nucleic acid sequence, wherein the duplex disrupts the repressive stem-loop structure to expose the RBS, thereby allowing translation of the gene.

2. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is not present, the cis-responsive nucleic acid sequence and RBS form a repressive stem-loop structure that blocks access of the ribosome to the RBS and represses translation of the gene by at least 80% as compared to translation of the gene when a repressive stem-loop structure is not formed.

3. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is not present, the cis-responsive nucleic acid sequence and RBS form a repressive stem-loop structure that blocks access of the ribosome to the RBS and represses translation of the gene by at least 90% as compared to translation of the gene when a repressive stem-loop structure is not formed ORF.

4. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is not present, the cis-responsive nucleic acid sequence and RBS form a repressive stem-loop structure that blocks access of the ribosome to the RBS and represses translation of the gene by at least 98% as compared to translation of the gene when a repressive stem-loop structure is not formed.

5. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is present, the trans-activating RNA and cis-responsive nucleic acid sequence form a duplex that disrupts the repressive stem-loop structure to expose the RBS to increase the translation of the gene by at least 5 fold relative to the level of translation when the trans-activating RNA is not present.

6. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is present, the trans-activating RNA and cis-repressive nucleic acid sequence form a duplex that disrupts the repressive stem-loop structure to expose the RBS to increase the translation of the gene by at least 10 fold relative to the level of translation when the trans-activating RNA is not present.

7. The recombinant prokaryotic cell of claim 1 , wherein when the trans-activating RNA is present, the trans-activating RNA and cis-repressive nucleic acid sequence form a duplex that disrupts the repressive stem-loop structure to expose the RBS to increase the translation of the gene by at least 19 fold relative to the level of translation when the trans-activating RNA is not present.

8. The recombinant prokaryotic cell of claim 1 , wherein the trans-activating nucleic acid sequence comprises, in 5′ to 3′ direction:
(i) a first non-stem forming portion, comprising at least 4 nucleotides that is complementary to, or substantially complementary to at least 4 nucleotides of the cis-repressive nucleic acid sequence,
(ii) a first stem-forming portion;
(iii) a second non-stem forming portion; and
(iv) a second stem-forming portion, comprising a nucleic acid sequence complementary to, or substantially complementary to the first-stem forming portion, wherein the first and second stem-forming portion complementary base pair to form a stem-loop structure, wherein the loop comprises the second non-stem forming portion,
wherein the first non-stem forming portion of the trans-activating nucleic acid sequence can form a duplex with at least 4 nucleotides of the cis-repressive nucleic acid sequence to disrupt the repressive stem-loop structure, thereby allowing translation of the gene.

9. The recombinant prokaryotic cell of claim 1 , wherein the loop of the repressive stem-loop structure comprises a YUNR sequence.

10. The recombinant prokaryotic cell of claim 1 , wherein the loop of the repressive stem-loop structure is 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.

11. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure is between 4 and 100 nucleotides in length, inclusive.

12. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure is between 6 and 50 nucleotides in length, inclusive.

13. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure is between 12 and 30 nucleotides in length, inclusive.

14. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure is approximately 19 nucleotides in length.

15. The recombinant prokaryotic cell of claim 8 , wherein the duplex of the repressive stem-loop structure comprises nucleic acid sequences that exhibit at least 66% complementarity.

16. The recombinant prokaryotic cell of claim 8 , wherein the duplex of the repressive stem-loop structure comprises nucleic acid sequences that exhibit between 75 and 95% complementarity.

17. The recombinant prokaryotic cell of claim 8 , wherein the duplex of the repressive stem-loop structure comprises nucleic acid sequences that exhibit approximately 85% complementarity.

18. The recombinant prokaryotic cell of claim 8 , wherein the duplex of the repressive stem-loop structure comprises at least one area of non-complementarity.

19. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure includes at least one bulge.

20. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure includes at least two dispersed areas of non-complementarity.

21. The recombinant prokaryotic cell of claim 20 , wherein the duplex of the repressive stem-loop structure includes at least two dispersed bulges.

22. The recombinant prokaryotic cell of claim 1 , wherein the duplex of the repressive stem-loop structure includes at

Assignees

• Collins James J
• Isaacs Farren J
• Cantor Charles R
• Dwyer Daniel J
• Univ Boston

Inventors

• Collins James J
• Isaacs Farren J
• Cantor Charles R
• Dwyer Daniel J

Classifications

• C12Q1/6897

  involving reporter genes operably linked to promoters · CPC title

• C12N2310/53

  partially self-complementary or closed · CPC title

• C12N15/11

  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

• C12N15/67 Primary

  General methods for enhancing the expression · CPC title