Accession Number : ADA501378


Title :   Riboswitch-Based Sensor in Low Optical Background


Descriptive Note : Conference paper


Corporate Author : HUMAN EFFECTIVENESS DIRECTORATE WRIGHT-PATTERSON AFB OH APPLIED BIOTECHNOLOGY BRANCH


Personal Author(s) : Harbaugh, Svetlana V ; Davidson, Molly E ; Chushak, Yaroslav G ; Kelley-Loughnane, Nancy ; Stone, Morley O


Full Text : http://www.dtic.mil/dtic/tr/fulltext/u2/a501378.pdf


Report Date : Aug 2008


Pagination or Media Count : 10


Abstract : Riboswitches are a type of natural genetic control element that use untranslated sequence in the RNA to recognize and bind to small molecules that regulate expression of that gene. Creation of synthetic riboswitches to novel ligands depends on the ability to screen for analyte binding sensitivity and specificity. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically-coded fluorescent proteins. Specifically, a theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence, and a FRET-based construct, BFP-eGFP or eGFP-REACh, was linked by a peptide encoding the recognition sequence for TEV protease. Cells expressing the riboswitch showed a marked optical difference in fluorescence emission in the presence of theophylline. However, the BFP-eGFP FRET pair posses significant optical background that reduces the sensitivity of a FRET-based assay. To improve the optical assay, we designed a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein) as the FRET acceptor for eGFP. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence which leads to an improved detection of FRET via better signal-to-noise ratio. The EGFP-REACh fusion protein was constructed with the TEV protease cleavage site; thus upon TEV translation, cleavage occurs diminishing REACh quenching and increasing eGFP emission resulting in a 4.5-fold improvement in assay sensitivity.


Descriptors :   *RIBONUCLEIC ACIDS , *FLUORESCENCE , GENE EXPRESSION , SPECTRUM ANALYZERS , PURINE ALKALOIDS , PROTEINS , SYMPOSIA , REGULATORS


Subject Categories : Genetic Engineering and Molecular Biology
      Test Facilities, Equipment and Methods
      Miscellaneous Detection and Detectors


Distribution Statement : APPROVED FOR PUBLIC RELEASE