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 . A regulatable gene expression construct comprising
 a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the riboswitch regulates expression of the sequence via dynamic interplay between the aptamer domain and the expression platform domain resulting in allosteric control of expression of the sequence, wherein the riboswitch and sequence are heterologous, wherein the sequence does not encode β-galactosidase.   
     
     
         2 . The construct of  claim 1  wherein the aptamer domain and the expression platform domain are heterologous. 
     
     
         3 . The construct of  claim 1  wherein the aptamer domain comprises a P1 stem, wherein the P1 stem comprises an aptamer strand and a control strand, wherein the expression platform domain comprises a regulated strand, wherein the regulated strand, the control strand, or both have been designed to form a stem structure. 
     
     
         4 . A riboswitch, wherein the riboswitch is a non-natural derivative of a naturally-occurring riboswitch, wherein the riboswitch is not derived from a naturally-occurring adenosylcobalamin-responsive riboswitch. 
     
     
         5 . The riboswitch of  claim 4  wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the aptamer domain and the expression platform domain are heterologous. 
     
     
         6 . The riboswitch of  claim 4  wherein the riboswitch is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch. 
     
     
         7 . The riboswitch of  claim 4  wherein the riboswitch is activated by a trigger molecule, wherein the riboswitch produces a signal when activated by the trigger molecule. 
     
     
         8 - 19 . (canceled) 
     
     
         20 . The construct of  claim 1 , wherein the expression platform domain comprises an expression regulatory element. 
     
     
         21 . The construct of  claim 20 , wherein the expression regulatory element is selected from the group comprising Shine-Dalgarno sequences, initiation codons, transcription terminators, and stability and processing signals. 
     
     
         22 . The construct of  claim 1 , wherein the aptamer domain does not control a ribozyme. 
     
     
         23 . The construct of  claim 1 , wherein the aptamer domain comprises a P1 stem, wherein the P1 stem comprises an aptamer strand and a control strand, wherein the expression platform domain comprises a regulated strand, wherein the regulated strand, the control strand, or both have been designed to form a stem structure,
 wherein the riboswitch is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch.   
     
     
         24 . The construct of  claim 23 , wherein the riboswitch is a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch. 
     
     
         25 . The construct of  claim 23 , wherein the derivative of the naturally-occurring riboswitch consists of only base pair conservative changes of the naturally-occurring riboswitch. 
     
     
         26 . The construct of  claim 1 , wherein the aptamer domain comprises a P1 stem, wherein the P1 stem comprises an aptamer strand and a control strand, wherein the expression platform domain comprises a regulated strand, wherein the regulated strand, the control strand, or both have been designed to form a stem structure,
 wherein the aptamer domain is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch.   
     
     
         27 . The construct of  claim 26 , wherein the aptamer domain is the aptamer domain of a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch. 
     
     
         28 . The construct of  claim 26 , wherein the derivative of the naturally-occurring riboswitch consists of only base pair conservative changes of the naturally-occurring riboswitch. 
     
     
         29 . The construct of  claim 1 , wherein the aptamer domain comprises a P1 stem, wherein the P1 stem comprises an aptamer strand and a control strand, wherein the expression platform domain comprises a regulated strand, wherein the regulated strand, the control strand, or both have been designed to form a stem structure,
 wherein the expression platform domain is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch.   
     
     
         30 . The construct of  claim 29 , wherein the expression platform domain is the expression platform domain of a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch. 
     
     
         31 . The construct of  claim 29 , wherein the derivative of the naturally-occurring riboswitch consists of only base pair conservative changes of the naturally-occurring riboswitch. 
     
     
         32 . The construct of  claim 1 , wherein the aptamer domain comprises a P1 stem, wherein the P1 stem comprises an aptamer strand and a control strand, wherein the expression platform domain comprises a regulated strand, wherein the regulated strand, the control strand, or both have been designed to form a stem structure,
 wherein the aptamer domain is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch,   wherein the expression platform domain is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch.   
     
     
         33 . The construct of  claim 32 , wherein the aptamer domain is the aptamer domain of a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch,
 wherein the expression platform domain is the expression platform domain of a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch.   
     
     
         34 . The construct of  claim 32 , wherein the derivative of the naturally-occurring riboswitch consists of only base pair conservative changes of the naturally-occurring riboswitch. 
     
     
         35 . The construct of  claim 1 , wherein the sequence comprises a coding region. 
     
     
         36 . The construct of  claim 1 , wherein the sequence encodes a protein or peptide of interest. 
     
     
         37 . The construct of  claim 1 , wherein the sequence comprises an expression product. 
     
     
         38 . The construct of  claim 1 , wherein the sequence is a heterologous sequence. 
     
     
         39 . The construct of  claim 1 , wherein binding of the aptamer domain by the trigger molecule results in allosteric control of expression of the sequence via interaction of the aptamer domain and the expression platform domain. 
     
     
         40 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the riboswitch regulates expression of the sequence, wherein the aptamer domain does not control a ribozyme, wherein the riboswitch and sequence are heterologous, wherein the sequence does not encode β-galactosidase. 
     
     
         41 . The construct of  claim 40 , wherein the riboswitch regulates expression of the sequence. 
     
     
         42 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence encoding a protein or peptide of interest, wherein the riboswitch regulates expression of the protein or peptide of interest, wherein the riboswitch and sequence encoding the protein or peptide of interest are heterologous, wherein the sequence does not encode β-galactosidase, wherein the riboswitch is derived from a naturally-occurring riboswitch. 
     
     
         43 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch regulates transcription, translation, or both transcription and translation of the sequence, wherein the riboswitch and sequence are heterologous, wherein the sequence does not encode β-galactosidase, wherein the riboswitch is derived from a naturally-occurring riboswitch. 
     
     
         44 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence encoding a protein or peptide of interest, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the riboswitch regulates expression of the sequence, wherein the riboswitch and the sequence encoding the protein or peptide of interest are heterologous, wherein the aptamer domain does not control a ribozyme, wherein the sequence does not encode β-galactosidase. 
     
     
         45 . The construct of  claim 44 , wherein the riboswitch regulates expression of the sequence. 
     
     
         46 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a coding region, wherein the coding region encodes a protein, wherein the riboswitch regulates expression of the protein, wherein the riboswitch and coding region are heterologous, wherein the coding region does not encode β-galactosidase, wherein the riboswitch is derived from a naturally-occurring riboswitch. 
     
     
         47 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a coding region, wherein the riboswitch regulates expression of the coding region, wherein the riboswitch and coding region are heterologous, wherein the coding region does not encode β-galactosidase, wherein the riboswitch is derived from a naturally-occurring riboswitch. 
     
     
         48 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch regulates expression of the sequence, wherein the riboswitch and sequence are heterologous, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the aptamer domain is a naturally-occurring aptamer, wherein the sequence does not encode β-galactosidase. 
     
     
         49 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch regulates expression of the sequence, wherein the riboswitch and sequence are heterologous, wherein the riboswitch is derived from a naturally-occurring guanine-responsive riboswitch, adenine-responsive riboswitch, lysine-responsive riboswitch, thiamine pyrophosphate-responsive riboswitch, adenosylcobalamin-responsive riboswitch, flavin mononucleotide-responsive riboswitch, or a S-adenosylmethionine-responsive riboswitch, wherein the derivative of the naturally-occurring riboswitch consists of only base pair conservative changes of the naturally-occurring riboswitch, wherein the sequence does not encode β-galactosidase. 
     
     
         50 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the aptamer changes state or structure when bound by a trigger molecule, wherein the expression platform domain changes state or structure when the aptamer domain changes state or structure, wherein the riboswitch regulates expression of the sequence, wherein the riboswitch and sequence are heterologous, wherein the sequence does not encode β-galactosidase. 
     
     
         51 . The construct of  claim 50 , wherein the change of state or structure of the expression platform domain comprises a change in base pairing of nucleotides in the expression platform domain. 
     
     
         52 . The construct of  claim 50 , wherein the change of state or structure of the aptamer domain comprises a change in base pairing of nucleotides in the aptamer domain upon binding the trigger molecule. 
     
     
         53 . The construct of  claim 50 , wherein the change of state or structure of the expression platform domain comprises formation of an alternative stem structure. 
     
     
         54 . The construct of  claim 50 , wherein binding of the aptamer domain by the trigger molecule results in allosteric control of expression of the sequence via interaction of the aptamer domain and the expression platform domain. 
     
     
         55 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the riboswitch regulates expression of the sequence via dynamic interplay between the aptamer domain and the expression platform domain resulting in allosteric control of expression of the sequence, wherein the aptamer domain and the expression platform domain are heterologous, wherein the sequence does not encode β-galactosidase. 
     
     
         56 . A regulatable gene expression construct comprising a nucleic acid molecule encoding an RNA comprising a riboswitch operably linked to a sequence, wherein the riboswitch comprises an aptamer domain and an expression platform domain, wherein the aptamer changes state or structure when bound by a trigger molecule, wherein the expression platform domain changes state or structure when the aptamer domain changes state or structure, wherein the riboswitch regulates expression of the sequence, wherein the aptamer domain and the expression platform domain are heterologous, wherein the sequence does not encode β-galactosidase.