Riboregulator compositions and methods of use

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.

1 . A toehold riboregulator comprising
 an RNA comprising
 a single-stranded toehold domain, 
 a fully or partially double-stranded stem domain comprising an initiation codon, 
 a loop domain comprising a ribosome binding site, and 
 a coding domain. 
   
     
     
         2 - 35 . (canceled) 
     
     
         36 . A beacon riboregulator comprising
 an RNA comprising
 a fully or partially double-stranded stem domain comprising a ribosome binding site, 
 a loop domain, and 
   a coding domain,   wherein an initiation codon is present between the stem domain and the coding domain.   
     
     
         37 . The riboregulator of  claim 36 , wherein the stem domain comprises sequence upstream (5′) of the initiation codon. 
     
     
         38 . (canceled) 
     
     
         39 . The riboregulator of  claim 36 , wherein the coding domain encodes a reporter protein. 
     
     
         40 . (canceled) 
     
     
         41 . The riboregulator of  claim 36 , wherein the coding domain encodes a non-reporter protein. 
     
     
         42 . The riboregulator of  claim 36 , wherein the loop domain is complementary in sequence to a naturally occurring RNA or a non-naturally occurring RNA. 
     
     
         43 . (canceled) 
     
     
         44 . A trans-activating RNA (taRNA) comprising
 a first domain that hybridizes to a loop domain of a riboregulator of  claim 36  and that comprises no or minimal secondary structure, and   a second domain that hybridizes to a sequence upstream (3′) of the loop domain and present in the stem domain.   
     
     
         45 . The trans-activating RNA of  claim 44 , wherein the first domain is 100% complementary to the loop domain. 
     
     
         46 . A system comprising
 the riboregulator of  claim 36 , and   a trans-activating RNA (taRNA) comprising a first domain that hybridizes to a loop domain of the riboregulator and that comprises no or minimal secondary structure, and a second domain that hybridizes to a sequence upstream (3′) of the loop domain and that is present in the stem domain.   
     
     
         47 . The system of  claim 46 , wherein the system is a cell or a cell-free in vitro system. 
     
     
         48 - 49 . (canceled) 
     
     
         50 . The system of  claim 46 , wherein the riboregulator and the taRNA are hybridized to each other. 
     
     
         51 . The system of  claim 46 , wherein the ratio of riboregulator to taRNA is less than 1, less than 0.5, or less than 0.1. 
     
     
         52 . The system of  claim 46 , wherein the riboregulator is comprised in a first nucleic acid and the taRNA is comprised in a second nucleic acid. 
     
     
         53 . The system of  claim 52 , wherein the first nucleic acid is a first plasmid and the second nucleic acid is a second plasmid. 
     
     
         54 . (canceled) 
     
     
         55 . A nucleic acid comprising
 the riboregulator of  claim 36 .   
     
     
         56 . A host cell comprising the nucleic acid of  claim 55 . 
     
     
         57 . A nucleic acid comprising
 the trans-activating RNA (taRNA) of  claim 44 .   
     
     
         58 . A host cell comprising the nucleic acid of  claim 57 . 
     
     
         59 . A method of detecting presence of an RNA in a sample, comprising
 combining a riboregulator of  claim 36  with a sample, wherein the riboregulator comprises a loop 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.   
     
     
         60 . A method of detecting presence of an RNA in a cell, comprising introducing into the cell a riboregulator of  claim 36 , wherein the riboregulator comprises a loop 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. 
     
     
         61 - 62 . (canceled) 
     
     
         63 . A method of controlling protein translation, comprising
 combining a riboregulator of  claim 36  with a taRNA comprising a first domain that hybridizes to a loop domain of the riboregulator and that comprises no or minimal secondary structure, and a second domain that hybridizes to a sequence upstream (3′) of the loop domain and that is present in the stem domain, wherein the riboregulator comprises a loop 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.