Compositions comprising riboregulators and methods of use thereof

Patent metadata

Field	Value
Publication number	US-11788156-B2
Application number	US-202117477456-A
Country	US
Kind code	B2
Filing date	Sep 16, 2021
Priority date	Jul 14, 2014
Publication date	Oct 17, 2023
Grant date	Oct 17, 2023

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 method of controlling gene and/or protein expression in a cell comprising
integrating or encoding a plurality of riboregulators into the genome of the cell, each riboregulator integrated upstream of a target coding sequence,
modulating expression of one or more of plurality of trans-activating RNA (taRNA) in the cell,
wherein expression of a taRNA in the cell results in increased expression of the target coding sequence, and
wherein each riboregulator comprises an RNA comprising
(i) a single-stranded toehold domain,
(ii) a fully or partially double-stranded stem domain comprising an initiation codon, and
(iii) a loop domain comprising a ribosome binding site, wherein each taRNA comprises
(i) a first domain that hybridizes to the toehold domain of one of the riboregulators in the plurality and that comprises no or minimal secondary structure, and
(ii) a second domain that hybridizes to a sequence downstream (3′) of the toehold domain.

2. The method of claim 1 , wherein expression of a plurality of target coding sequences is controlled.

3. The method of claim 2 , wherein the plurality of target coding sequences encode proteins that interact with each other directly or indirectly.

4. The method of claim 1 , wherein the plurality of taRNA are integrated or encoded in the host cell genome.

5. The method of claim 1 , wherein each taRNA is operably linked to an inducible promoter that is different from all the other taRNA in the plurality.

6. The method of claim 1 , wherein each taRNA has a cognate riboregulator in the cell.

7. The method of claim 1 , wherein at least one taRNA activates two or more riboregulators in the cell.

8. A method of controlling gene and/or protein expression in a cell comprising
introducing a plurality of riboregulators into a cell, each riboregulator comprises an RNA comprising codon,
(i) a single-stranded toehold domain,
(ii) a fully or partially double-stranded stem domain comprising an initiation codon,
(iii) a loop domain comprising a ribosome binding site, and
(iv) a coding domain for a reporter protein or a protein of interest, modulating expression of one or more of a plurality of trans-activating RNA (taRNA) in the cell,

wherein expression of a taRNA in the cell results in increased expression of the coding domain, and
wherein each taRNA comprises
(i) a first domain that hybridizes to the toehold domain of one of the riboregulators in the plurality and that comprises no or minimal secondary structure, and
(ii) a second domain that hybridizes to a sequence downstream (3′) of the toehold domain.

9. The method of claim 8 , wherein one or more riboregulators comprise a coding domain for a transcription factor.

10. The method of claim 8 , wherein modulating expression of one or more of a plurality of taRNA comprises increasing expression of a subset of taRNA substantially simultaneously.

Assignees

• Harvard College
• Univ Boston

Inventors

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

Classifications

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title

• 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

• C12N15/1138

  against receptors or cell surface proteins · CPC title