Characterization of a riboswitch for detection of the insensitive munition hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX).

RDX is a commonly used munition that is of significant environmental concern due to manufacturing and training activities that have led to wide scale contamination. Detecting RDX residues in the environment is difficult since it is typically present in low concentrations. One promising approach that has been used for small molecule detection is aptamer based sensors. The focus of this research effort was to isolate and characterize aptamers capable of binding RDX and incorporate these aptamers as riboswitches to regulate expression of a fluorescence gene.

Aptamers were selected by systematic evolution of ligands by exponential enrichment (SELEX) and sequences with high affinity for RDX were cloned up stream of the DsRed-Express2 fluorescence gene with a 6 nucleotide random linker sequence between the aptamer and DsRed-Express2. A clone library of random linker sequences was screened for individual clones that showed increased levels of fluorescence in the presence of 10 ppm RDX. The greatest response was with clone 11 which showed an 11-fold increase in fluorescence in the presence of 10 ppm RDX. Cultures exposed to RDX had visibly distinct red color compared to controls. Clones with a 2-fold or greater increase in fluorescence in the presence of RDX were further characterized over a range of RDX concentrations. The results of this assay showed an increase in fluorescence response between 0-1 ppm RDX. Above 1 ppm RDX the fluorescence response was saturated. This work demonstrated that an RDX riboswitch regulated fluorescence gene could function as a biosensor for detection of RDX.