Sensitive and Specific Detection of Ligands Using Engineered Riboswitches

Riboswitches are RNA elements found in non‐coding regions of messenger RNAs that regulate gene expression through a ligand‐triggered conformational change (for review, see ref. 1). In, B. subtilis and other gram positive bacteria , purine metabolism is regulated by riboswitches that bind to guanine (2). The best studied of the guanine riboswitches regulates the transcription of the B. subtilis xpt‐pbuX operon. Guanine binding to this riboswitch inhibits transcription by stabilizing an alternative conformation that allows formation of a premature transcriptional terminator. In order to study guanine‐triggered structure switching, we have developed a fluorescence quenching assay for the function of the guanine riboswitch. Our system consists of the guanine‐binding domain (aptamer domain) of the riboswitch (xpt RNA), and two DNA oligonucleotides that are related to the 5′ and 3′ halves of the terminator stem (5′T and 3′T). xpt RNA is labeled at its 3′ end with a fluorophore and 5′T is labeled at its 5′ end with a fluorescence quencher. When 5′T is annealed to a complementary sequence at the 3′ end of xpt RNA, fluorescence is quenched. Guanine binding causes 5′T to dissociate from xpt RNA and anneal to 3′T. This strand‐exchange reaction causes an increase in fluorescence intensity as the quencher moves away from the fluorophore. Using this assay, we reproducibly detected as little as 5 nM guanine. We are currently using the same strategy to adapt a 2′‐deoxyguanosine riboswitch (3) for use as a probe that can detect its ligand in vitro . In addition, we have used our strand‐exchange reaction to develop a method for in vitro selection of variant riboswitches with altered ligand specificity. The results of our latest experiments will be discussed. Support or Funding Information Defense Threat Reduction Agency (DTRA), Office of Naval Research (ONR), Chemistry Department, U.S, Naval Academy