Caged RNAs and methods of use thereof

Abstract

Methods of using labeled interfering RNAs to detect and/or quantitate target mRNAs in cells are provided. Related compositions, systems, and kits are also provided. Caged interfering RNAs (e.g., photoactivatable interfering RNAs), methods of using such caged RNAs, and related systems and kits are also provided. Caged RNAs capable of repressing translation of a target mRNA or silencing transcription of a target gene are also provided, along with related methods, systems, and kits. Methods and compositions for introducing RNAs into cells, using RNAs covalently associated with protein transduction domains and/or lipids, are provided. Also provided are methods and compositions for selectively attenuating expression of a target mRNA by controlling expression of an interfering RNA, an RNA capable of initiating translation repression, or an RNA capable of initiating transcriptional silencing.

1 . A composition comprising a caged RNA, the caged RNA comprising: 
 an RNA capable of repressing translation of a target mRNA; and,    one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from repressing translation of the target mRNA in a cell comprising the caged RNA.    
     
     
         2 . The composition of  claim 1 , wherein the RNA does not initiate degradation of the target mRNA in a cell comprising the RNA.  
     
     
         3 . The composition of  claim 1 , wherein the RNA is double-stranded.  
     
     
         4 . The composition of  claim 1 , wherein the RNA comprises at least an antisense strand, the antisense strand comprising a first region which is complementary to a second region of the target mRNA, the first region being interrupted by one or more nucleotides which are not complementary to the second region.  
     
     
         5 . The composition of  claim 4 , wherein the first region is interrupted by two, three, four, or more nucleotides which are not complementary to the second region.  
     
     
         6 . The composition of  claim 4 , wherein the second region is within the 3′-untranslated region of the target mRNA.  
     
     
         7 . The composition of  claim 4 , wherein the RNA comprises at least one double-stranded region, the double-stranded region comprising the antisense strand and a sense strand.  
     
     
         8 . The composition of  claim 7 , wherein the sense strand is completely complementary to the antisense strand over the double-stranded region.  
     
     
         9 . The composition of  claim 7 , wherein the sense strand is not completely complementary to the antisense strand over the double-stranded region.  
     
     
         10 . The composition of  claim 7 , wherein the RNA comprises a first polyribonucleotide comprising the sense strand and a second polyribonucleotide comprising the antisense strand.  
     
     
         11 . The composition of  claim 10 , wherein the first polyribonucleotide comprises between 17 and 29 nucleotides, the second polyribonucleotide comprises between 17 and 29 nucleotides, and the double-stranded region comprises between 17 and 29 base pairs.  
     
     
         12 . The composition of  claim 11 , wherein the first polyribonucleotide comprises between 18 and 25 nucleotides, the second polyribonucleotide comprises between 18 and 25 nucleotides, and the double-stranded region comprises between 18 and 25 base pairs.  
     
     
         13 . The composition of  claim 11 , wherein the first polyribonucleotide and the second polyribonucleotide each comprise a two nucleotide TT 3′ overhang.  
     
     
         14 . The composition of  claim 10 , wherein at least one of the one or more first caging groups is covalently attached to a 5′ hydroxyl or a 5′ phosphate of the second polyribonucleotide.  
     
     
         15 . The composition of  claim 10 , wherein the first caging group is covalently attached to the first polyribonucleotide and to the second polyribonucleotide.  
     
     
         16 . The composition of  claim 15 , wherein the first caging group is attached to the 3′ end of the first polyribonucleotide and to the 5′ end of the second polyribonucleotide.  
     
     
         17 . The composition of  claim 7 , wherein the RNA comprises a self-complementary polyribonucleotide.  
     
     
         18 . The composition of  claim 1 , comprising the target mRNA, a cell, a cell comprising the target mRNA, or a cell comprising the caged RNA.  
     
     
         19 . The composition of  claim 1 , wherein the first caging groups inhibit the RNA from repressing translation of the target mRNA by at least about 30%, at least about 50%, at least about 75%, at least about 90%, at least about 95%, or at least about 98%, as compared to the RNA in the absence of the first caging groups.  
     
     
         20 . The composition of  claim 1 , wherein the first caging groups prevent the RNA from repressing translation of the target mRNA.  
     
     
         21 . The composition of  claim 1 , wherein removal of or an induced conformational change in the first caging groups permits the RNA to repress translation of the target mRNA.  
     
     
         22 . The composition of  claim 1 , wherein the one or more first caging groups associated with the RNA are covalently attached to the RNA.  
     
     
         23 . The composition of  claim 1 , wherein the one or more first caging groups are removable by sonication, photoactivatable, or photolabile.  
     
     
         24 . The composition of  claim 1 , wherein the one or more first caging groups each comprises a first binding moiety; the composition comprising a second binding moiety that can bind at least one of the first binding moieties.  
     
     
         25 . The composition of  claim 1 , wherein the RNA comprises at least one label.  
     
     
         26 . The composition of  claim 1 , wherein the RNA is associated with a cellular delivery module that can mediate introduction of the RNA into a cell.  
     
     
         27 . The composition of  claim 26 , wherein the cellular delivery module comprises a polypeptide, a PEP-1 pep tide, an amphipathic peptide, an MPG ΔNLS  peptide, a cationic peptide, a homopolymer of D-arginine, a homopolymer of histidine, a homopolymer of lysine, a protein transduction domain, a protein transduction domain derived from an HIV-1 Tat protein, from a herpes simplex virus VP22 protein, or from a  Drosophila antennapedia  protein, a model protein transduction domain, or a model protein transduction domain comprising a homopolymer of D-arginine.  
     
     
         28 . The composition of  claim 26 , wherein the cellular delivery module is covalently attached to the RNA.  
     
     
         29 . The composition of  claim 28 , wherein the cellular delivery module is attached to the RNA through a disulfide bond, or wherein the covalent attachment is reversible by exposure to light of a preselected wavelength.  
     
     
         30 . The composition of  claim 28 , wherein the cellular delivery module comprises a lipid or one or more myristoyl groups.  
     
     
         31 . The composition of  claim 26 , wherein the cellular delivery module is associated with one or more second caging groups which inhibit the cellular delivery module from mediating introduction of the RNA into a cell.  
     
     
         32 . The composition of  claim 1 , wherein the RNA comprises a first polyribonucleotide comprising a sense strand and a second polyribonucleotide comprising an antisense strand, and wherein a cellular delivery module is covalently attached to the second polyribonucleotide.  
     
     
         33 . The composition of  claim 1 , wherein the first caging group is a cellular delivery module.  
     
     
         34 . The composition of  claim 1 , wherein the caged RNA is bound to a matrix.  
     
     
         35 . The composition of  claim 34 , wherein the matrix is a surface, and the RNA is bound to the surface at a predetermined location within an array comprising other RNAs.  
     
     
         36 . A kit for making the caged RNA of  claim 1 , comprising an RNA, one or more first caging groups, and instructions for assembling the RNA and the first caging groups to form the caged RNA, packaged in one or more containers; or comprising one or more first caging groups and instructions for assembling the first caging groups and an RNA supplied by a user of the kit to form the caged RNA, packaged in one or more containers.  
     
     
         37 . A method of selectively attenuating expression of a target gene in a cell, the method comprising: 
 introducing a caged RNA into the cell, the caged RNA comprising 
 (a) an RNA capable of repressing translation of a target mRNA transcribed from the target gene, and  
 (b) one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from repressing translation of the target mRNA in the cell; and,  
   initiating repression of translation of the target mRNA by exposing the cell to uncaging energy, whereby exposure to the uncaging energy frees the RNA from inhibition by the caging groups.    
     
     
         38 . The method of  claim 37 , wherein the amount of the target mRNA present in the cell is not affected by the presence of the RNA in the cell.  
     
     
         39 . The method of  claim 37 , wherein exposing the cell to uncaging energy comprises exposing the cell to light of a first wavelength.  
     
     
         40 . The method of  claim 39 , wherein exposing the cell to light of the first wavelength comprises exposing the cell to light wherein intensity of the light and duration of exposure of the cell to the light are controlled such that a first portion of the caged RNA is uncaged and a second portion of the caged RNA remains caged.  
     
     
         41 . The method of  claim 40 , comprising exposing the cell to light of the first wavelength again.  
     
     
         42 . The method of  claim 40 , wherein the first portion is a selected amount.  
     
     
         43 . The method of  claim 37 , comprising contacting the cell and a test compound, and wherein the cell is exposed to the uncaging energy at a preselected time point with respect to a time at which the cell and the test compound are contacted.  
     
     
         44 . The method of  claim 37 , wherein the uncaging energy is directed at a preselected subset of a cell population comprising the cell.  
     
     
         45 . The method of  claim 37 , wherein the caged RNA comprises a cellular delivery module that can mediate introduction of the caged RNA into the cell, the cellular delivery module being associated with the RNA, and wherein introducing the caged RNA into the cell comprises contacting the cell with the caged RNA associated with the cellular delivery module.  
     
     
         46 . The method of  claim 37 , wherein the RNA comprises at least one label, the method comprising detecting a signal from the label.  
     
     
         47 . A composition comprising a caged RNA, the caged RNA comprising: 
 an RNA capable of silencing transcription of a target gene; and,    one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from silencing transcription of the target gene in a cell comprising the caged RNA.    
     
     
         48 . A method of selectively attenuating expression of a target gene in a cell, the method comprising: 
 introducing a caged RNA into the cell, the caged RNA comprising 
 (a) an RNA capable of silencing transcription of the target gene, and  
 (b) one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from silencing transcription of the target gene in the cell; and,  
   initiating silencing of transcription of the target gene by exposing the cell to uncaging energy, whereby exposure to the uncaging energy frees the RNA from inhibition by the caging groups.    
     
     
         49 . A method of selectively attenuating expression of a target gene in a cell, the method comprising: 
 introducing a first caged DNA and a second caged DNA into the cell, the first caged DNA comprising a first DNA encoding an RNA sense strand and one or more caging groups associated with the first DNA, the second caged DNA comprising a second DNA encoding an RNA antisense strand and one or more caging groups associated with the second DNA, the caging groups inhibiting transcription of the first and second DNAs, the first and second DNAs each comprising at least a portion of the target gene, and the sense and antisense strands being at least partially complementary and able to form a duplex over at least a portion of their lengths; and,    initiating translational repression by generating double-stranded RNA by exposing the cell to uncaging energy, whereby exposure to the uncaging energy frees the first and second DNAs from inhibition by the caging groups and permits transcription of the first and second DNAs to occur.    
     
     
         50 . The method of  claim 49 , wherein the sense strand comprises a first polyribonucleotide and the antisense strand comprises a second polyribonucleotide, or wherein the sense and antisense strands comprise a single, self-complementary polyribonucleotide.  
     
     
         51 . The method of  claim 49 , wherein exposing the cell to uncaging energy comprises exposing the cell to light of a first wavelength.  
     
     
         52 . A composition, comprising: 
 a protein transduction domain covalently attached to an RNA; and,    the RNA, which RNA comprises: 
 (a) at least one double-stranded region, the double-stranded region comprising a sense strand and an antisense strand, the antisense strand comprising a region which is substantially complementary to a region of a target mRNA, or  
 (b) a single polyribonucleotide strand comprising an antisense strand, the antisense strand comprising a region which is substantially complementary to a region of a target mRNA corresponding to the target gene.  
   
     
     
         53 . The composition of  claim 52 , wherein the region of the antisense strand is completely complementary to the region of the target mRNA.  
     
     
         54 . The composition of  claim 52 , wherein the region of the antisense strand which is substantially complementary to the region of the target mRNA comprises at least a first and a second subregion, each of which is completely complementary to the target mRNA, flanking one or more nucleotides which are not complementary to the target mRNA.  
     
     
         55 . The composition of  claim 54 , wherein the first and second subregions flank two, three, four, or more nucleotides which are not complementary to the target mRNA.  
     
     
         56 . The composition of  claim 52 , comprising one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from repressing translation of the target mRNA in a cell.  
     
     
         57 . A method of introducing an RNA into a cell, the method comprising: 
 (a) providing a composition comprising 
 (i) an RNA comprising at least one double-stranded region, the double-stranded region comprising a sense strand and an antisense strand, the antisense strand comprising a region which is substantially complementary to a region of a target mRNA; or an RNA comprising a single polyribonucleotide strand comprising an antisense strand, the antisense strand comprising a region which is substantially complementary to a region of a target mRNA, and  
 (ii) a protein transduction domain covalently attached to the RNA; and,  
   (b) contacting the composition and the cell, whereby the protein transduction domain mediates introduction of the RNA into the cell.    
     
     
         58 . The method of  claim 57 , wherein the composition comprises one or more first caging groups associated with the RNA, the first caging groups inhibiting the RNA from repressing translation of the target mRNA in the cell; the method comprising initiating translational repression of the target mRNA by exposing the cell to uncaging energy of a first type, whereby exposure to the uncaging energy frees the RNA from inhibition by the first caging groups.