Riboswitches, methods for their use, and compositions for use with riboswitches

Abstract

It has been discovered that certain natural mRNAs serve as metabolite-sensitive genetic switches wherein the RNA directly binds a small organic molecule. This binding process changes the conformation of the mRNA, which causes a change in gene expression by a variety of different mechanisms. Modified versions of these natural “riboswitches” (created by using various nucleic acid engineering strategies) can be employed as designer genetic switches that are controlled by specific effector compounds. Such effector compounds that activate a riboswitch are referred to herein as trigger molecules. The natural switches are targets for antibiotics and other small molecule therapies. In addition, the architecture of riboswitches allows actual pieces of the natural switches to be used to construct new non-immunogenic genetic control elements, for example the aptamer (molecular recognition) domain can be swapped with other non-natural aptamers (or otherwise modified) such that the new recognition domain causes genetic modulation with user-defined effector compounds. The changed switches become part of a therapy regimen-turning on, or off, or regulating protein synthesis. Newly constructed genetic regulation networks can be applied in such areas as living biosensors, metabolic engineering of organisms, and in advanced forms of gene therapy treatments.

1 - 7 . (canceled) 
     
     
         8 . A method of detecting a compound of interest, the method comprising bringing into contact a sample and a riboswitch, wherein the riboswitch is activated by the compound of interest, wherein the riboswitch produces a signal when activated by the compound of interest, wherein the riboswitch produces a signal when the sample contains the compound of interest. 
     
     
         9 . The method of  claim 8  wherein the riboswitch changes conformation when activated by the compound of interest, wherein the change in conformation produces a signal via a conformation dependent label. 
     
     
         10 . The method of  claim 8  wherein the riboswitch changes conformation when activated by the compound of interest, wherein the change in conformation causes a change in expression of an RNA linked to the riboswitch, wherein the change in expression produces a signal. 
     
     
         11 . The method of  claim 10  wherein the signal is produced by a reporter protein expressed from the RNA linked to the riboswitch. 
     
     
         12 . A method of inhibiting gene expression, the method comprising bringing into contact a compound and a cell, 
       wherein the compound has the structure 
       
         
           
           
               
               
           
         
         wherein, when the compound is bound to a guanine-responsive riboswitch, R 7  serves as a hydrogen bond acceptor, R 10  serves as a hydrogen bond donor, R 11  serves as a hydrogen bond acceptor, R 12  serves as a hydrogen bond donor, 
         wherein R 13  is H, H 2  or is not present, 
         wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , and R 9  are each independently C, N, O, or S, 
         wherein   each independently represent a single or double bond, 
         wherein the compound is not guanine, hypoxanthine, or xanthine, 
         wherein the cell comprises a gene encoding an RNA comprising a guanine-responsive riboswitch, wherein the compound inhibits expression of the gene by binding to the guanine-responsive riboswitch. 
       
     
     
         13 . A method of inhibiting gene expression, the method comprising bringing into contact a compound and a cell,
 wherein the compound has the structure   
       
         
           
           
               
               
           
         
         wherein, when the compound is bound to an adenine-responsive riboswitch, R 1 , R 3  and R 7  serve as hydrogen bond acceptors, and R 10  and R 11  serve as hydrogen bond donors, 
         wherein R 12  is H, H 2  or is not present, 
         wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 8 , and R 9  are each independently C, N, O, or S, 
         wherein   each independently represent a single or double bond, 
         wherein the compound is not adenine, 2,6-diaminopurine, or 2-amino purine, 
         wherein the cell comprises a gene encoding an RNA comprising an adenine-responsive riboswitch, wherein the compound inhibits expression of the gene by binding to the adenine-responsive riboswitch. 
       
     
     
         14 . A method of inhibiting gene expression, the method comprising bringing into contact a compound and a cell,
 wherein the compound has the structure   
       
         
           
           
               
               
           
         
         wherein R 2  and R 3  are each positively charged, 
         wherein R 1  is negatively charged, 
         wherein R 4  is C, N, O, or S, 
         wherein   each independently represent a single or double bond, 
         wherein the compound is not lysine, 
         wherein the cell comprises a gene encoding an RNA comprising a lysine-responsive riboswitch, wherein the compound inhibits expression of the gene by binding to the lysine-responsive riboswitch. 
       
     
     
         15 . The method of  claim 14  wherein R 2  and R 3  are each NH 3   +  and wherein R 1  is O − . 
     
     
         16 . A method of inhibiting gene expression, the method comprising bringing into contact a compound and a cell,
 wherein the compound has the structure   
       
         
           
           
               
               
           
         
         wherein R 1  is positively charged, 
         wherein R 2  and R 3  are each independently C, O, or S, 
         wherein R 4  is CH 3 , NH 2 , OH, SH, H or not present, 
         wherein R 5  is CH 3 , NH 2 , OH, SH, or H, 
         wherein R 6  is C or N, 
         wherein   each independently represent a single or double bond, 
         wherein the compound is not TPP, TP or thiamine, 
         wherein the cell comprises a gene encoding an RNA comprising a thiamine pyrophosphate-responsive riboswitch, wherein the compound inhibits expression of the gene by binding to the thiamine pyrophosphate-responsive riboswitch. 
       
     
     
         17 . The method of  claim 16  wherein R 1  is phosphate, diphosphate or triphosphate. 
     
     
         18 . A method comprising
 (a) testing a compound for inhibition of gene expression of a gene encoding an RNA comprising a riboswitch, wherein the inhibition is via the riboswitch,   (b) inhibiting gene expression by bringing into contact a cell and a compound that inhibited gene expression in step (a),   wherein the cell comprises a gene encoding an RNA comprising a riboswitch, wherein the compound inhibits expression of the gene by binding to the riboswitch.   
     
     
         19 . A method of identifying riboswitches, the method comprising
 assessing in-line spontaneous cleavage of an RNA molecule in the presence and absence of a compound, wherein the RNA molecule is encoded by a gene regulated by the compound, and   assessing the expression of the RNA molecule in the presence and absence of the compound,   wherein both a change in the pattern of in-line spontaneous cleavage of the RNA molecule and a change in the expression of the RNA molecule indicates a riboswitch.   
     
     
         20 . The method of  claim 12 , wherein the cell is killed or growth of the cell is inhibited. 
     
     
         21 . The method of  claim 12 , wherein the cell is a bacterial cell. 
     
     
         22 . The method of  claim 12 , wherein the cell is in a patient. 
     
     
         23 . The method of  claim 12 , wherein the cell is a bacterial cell, wherein the cell is in a patient. 
     
     
         24 . The method of  claim 13 , wherein the cell is killed or growth of the cell is inhibited. 
     
     
         25 . The method of  claim 13 , wherein the cell is a bacterial cell. 
     
     
         26 . The method of  claim 13 , wherein the cell is in a patient. 
     
     
         27 . The method of  claim 13 , wherein the cell is a bacterial cell, wherein the cell is in a patient. 
     
     
         28 . The method of  claim 14 , wherein the cell is killed or growth of the cell is inhibited. 
     
     
         29 . The method of  claim 14 , wherein the cell is a bacterial cell. 
     
     
         30 . The method of  claim 14 , wherein the cell is in a patient. 
     
     
         31 . The method of  claim 14 , wherein the cell is a bacterial cell, wherein the cell is in a patient. 
     
     
         32 . The method of  claim 16 , wherein the cell is killed or growth of the cell is inhibited. 
     
     
         33 . The method of  claim 16 , wherein the cell is a bacterial cell. 
     
     
         34 . The method of  claim 16 , wherein the cell is in a patient. 
     
     
         35 . The method of  claim 16 , wherein the cell is a bacterial cell, wherein the cell is in a patient. 
     
     
         36 . The method of  claim 16 , wherein the cell is a fungal cell. 
     
     
         37 . The method of  claim 16 , wherein the cell is a plant cell. 
     
     
         38 . The method of  claim 18 , wherein the cell is killed or growth of the cell is inhibited. 
     
     
         39 . The method of  claim 18 , wherein the cell is a bacterial cell. 
     
     
         40 . The method of  claim 18 , wherein the cell is in a patient. 
     
     
         41 . The method of  claim 18 , wherein the cell is a bacterial cell, wherein the cell is in a patient. 
     
     
         42 . The method of  claim 8 , wherein detection of the compound of interest identifies the compound of interest as a compound that activates the riboswitch. 
     
     
         43 . The method of  claim 42 , wherein activation of the riboswitch inhibits expression of a gene encoding an RNA comprising the riboswitch. 
     
     
         44 . The method of  claim 42 , wherein activation of the riboswitch induces expression of a gene encoding an RNA comprising the riboswitch. 
     
     
         45 . The method of  claim 10 , wherein the signal is produced by labeled nucleotides incorporated into the expressed RNA linked to the riboswitch. 
     
     
         46 . The method of  claim 10 , wherein the change in expression of the RNA linked to the riboswitch is inhibition of expression. 
     
     
         47 . The method of  claim 10 , wherein the change in expression of the RNA linked to the riboswitch is induction of expression. 
     
     
         48 . A method comprising
 testing a compound for inhibition of expression of a gene encoding an RNA comprising a riboswitch, wherein the inhibition is via the riboswitch, wherein if the compound inhibits expression of the gene encoding the RNA comprising the riboswitch then the compound is identified as a trigger molecule of the riboswitch, wherein the compound inhibits expression of the gene by binding to the riboswitch.   
     
     
         49 . A method of killing or inhibiting the growth of a bacterial cell, the method comprising bringing into contact the cell and a compound identified by the method of  claim 48 , wherein the growth of the cell is inhibited. 
     
     
         50 . The method of  claim 49 , wherein the cell is in a patient. 
     
     
         51 . A method of killing or inhibiting the growth of a fungal cell, the method comprising bringing into contact the cell and a compound identified by the method of  claim 48  wherein the growth of the cell is inhibited. 
     
     
         52 . The method of  claim 51 , wherein the cell is in a patient. 
     
     
         53 . A method of inhibiting cell growth, the method comprising bringing into contact a cell and a compound, wherein the compound can bind to a riboswitch, wherein the cell comprises a gene encoding an RNA comprising a riboswitch, wherein the compound inhibits expression of the gene by binding to the riboswitch. 
     
     
         54 . The method of  claim 53 , wherein the riboswitch is a guanine-responsive riboswitch. 
     
     
         55 . The method of  claim 53 , wherein the riboswitch is an adenine-responsive riboswitch. 
     
     
         56 . The method of  claim 53 , wherein the riboswitch is a lysine-responsive riboswitch. 
     
     
         57 . The method of  claim 53 , wherein the riboswitch is a thiamine pyrophosphate-responsive riboswitch. 
     
     
         58 . The method of  claim 53 , wherein the riboswitch is a flavin mononucleotide-responsive riboswitch.