Compositions comprising riboregulators and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11124846-B2
Application number	US-201515326054-A
Country	US
Kind code	B2
Filing date	Jul 14, 2015
Priority date	Jul 14, 2014
Publication date	Sep 21, 2021
Grant date	Sep 21, 2021

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 system comprising
a host cell having, or encoded in its genome, an n-OR riboregulator system having n number of AND gates arranged 5′ to 3′, wherein n is at least 4;
wherein an AND gate comprises, in a 5′ to 3′ order:
(i) a single-stranded toehold domain, and
(ii) a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain and at least two binding domains;

wherein the loop domain comprises a ribosome binding site;
wherein the AND gates are upstream of a coding sequence,
wherein the AND gate that is most 5′ in the n-OR riboregulator system has an ON/OFF ratio that is higher than any one of the other AND gates in the n-OR riboregulator system, wherein the ON/OFF ratio is ratio of expression from an AND gate in the presence and absence of cognate inputs, and
wherein expression of the coding sequence is controlled by the n-OR riboregulator system.

2. The system of claim 1 , wherein the host cell is a prokaryotic cell.

3. The system of claim 1 , wherein the host cell is a bacterial cell.

4. The system of claim 1 , wherein the host cell is an E. coli bacterium.

5. The system of claim 1 , wherein the AND gates within the n-OR riboregulator system are separated from each other by 0-30 nucleotides, or 9-15 nucleotides.

6. The system of claim 1 , wherein the n-OR riboregulator system further comprises a spacer domain that
(i) is situated between the most 3′ AND gate and the coding sequence, or
(ii) is situated between two adjacent AND gates.

7. The system of claim 6 , wherein the spacer domain is about 9-33 nucleotides in length, or about 21 nucleotides in length.

8. The system of claim 1 , wherein the initiation codon is wholly or partially present in a 1-3 nucleotide bulge in the stem domain.

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

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

11. The system of claim 9 , wherein the sequence downstream of the initiation codon
(a) is about 9 nucleotides, and/or
(b) does not encode a stop codon.

12. The system of claim 1 , wherein the coding sequence encodes a reporter protein.

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

14. The system of claim 1 , wherein the coding sequence encodes a non-reporter protein.

15. The system of claim 1 , wherein the toehold domain is
(a) complementary in sequence to a naturally occurring RNA, or
(b) complementary in sequence to a non-naturally occurring RNA.

16. The system of claim 1 , further comprising
a plurality of trans-activating RNAs (taRNAs), each comprising
(i) a first domain that hybridizes to the toehold domain of one of the AND gates or to another trans-activating RNA (taRNA) and that comprises no or minimal secondary structure, and
(ii) a second domain that hybridizes to one of the at least two binding domains in the AND gate

wherein each taRNA has a cognate binding domain,
wherein hybridization of taRNAs to the at least two binding domains on an AND gate activates the AND gate, and
wherein activation of any one of the AND gates is sufficient to activate expression of the coding sequence.

17. A method of detecting presence of a plurality of RNA in a sample, comprising
combining a plurality of n-OR riboregulator systems with a sample, wherein each n-OR riboregulator system comprises n number of AND gates arranged 5′ to 3′, wherein n is at least 4;
wherein an AND gate comprises, in a 5′ to 3′ order:
(i) a single-stranded toehold domain, and
(ii) a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain and at least two binding domains;

wherein the loop domain comprises a ribosome binding site,
wherein the AND gates are upstream to a coding sequence,
wherein the AND gate that is most 5′ in the n-OR riboregulator system has an ON/OFF ratio that is higher than any one of the other AND gates in the n-OR riboregulator system, wherein the ON/OFF ratio is ratio of expression from an AND gate in the presence and absence of cognate inputs, and
wherein the coding sequence encodes a reporter protein, under conditions that allow translation of the coding sequence in the presence of endogenous RNA that binds to all of the binding domains on any one of the AND gates; and
detecting the reporter protein as an indicator of the endogenous RNA,
wherein each n-OR riboregulator system detects different endogenous RNA from all other n-OR riboregulator systems in the plurality, and each n-OR riboregulator system encodes a different reporter protein from all other n-OR riboregulator systems in the plurality.

18. A method of detecting presence of a plurality of RNA in a cell, comprising
introducing into the cell a plurality of n-OR riboregulator systems, wherein each n-OR riboregulator system comprises n number of AND gates arranged 5′ to 3′, wherein n is at least 4;
wherein an AND gate comprises in a 5′ to 3′ order:
(i) a single-stranded toehold domain, and
(ii) a fully or partially double-stranded stem domain comprising an initiation codon, a loop domain and at least two binding domains;

wherein the loop domain comprises a ribosome binding site,
wherein the AND gates are upstream to a coding sequence,
wherein the AND gate that is most 5′ in the n-OR riboregulator system has an ON/OFF ratio that is higher than any one of the other AND gates in the n-OR riboregulator system, wherein the ON/OFF ratio is ratio of expression from an AND gate in the presence and absence of cognate inputs, and
wherein the coding sequence encodes a reporter protein, under conditions that allow translation of the coding sequence in the presence of endogenous RNA that binds to all of the binding domains on any one of the AND gates; and
detecting the reporter protein as an indicator of the endogenous RNA,
wherein each n-OR riboregulator system detects different endogenous RNA from all other n-OR riboregulator systems in the plurality, and each n-OR riboregulator system encodes a different reporter protein from all other n-OR riboregulator systems in the plurality.

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

20. The system of claim 18 , wherein the host cell is a bacterial cell.

21. The system of claim 18 , wherein the host cell is an E. coli bacterium.

Assignees

• Harvard College
• Univ Boston

Inventors

• Green Alexander A
• Yin Peng
• Collins James J
• Kim Jongmin

Classifications

• C12N15/113 Primary

  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 title

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title

• C12N15/1138

  against receptors or cell surface proteins · CPC title