RNA plays an important role in the regulation of gene expression. This function is intimately related to its structure. Currently, only a few methods exist to study RNA structures in vivo. In our previous work, we explored the use of artificial translational regulators to link expression of a fluorescent gene to the detection of �accessible� structural regions within an RNA molecule. We engineered a riboswitch containing a probe complementary to a specific sequence on the target RNA molecule, a stem-loop structure consists of a cis-blocking (cb) element sequestering the complementary RBS site, and GFP as a reporter. The expression of GFP is correlated to the local structural changes of the riboswitch and is dependent on the location of the region on the target molecule we are probing. At this point, it is not well understood how different parameters of these probes affect their function. The length of the probe is an important aspect to consider because it has the possibility of maximizing the dynamic range of our fluorescence assay. We present the effect of probe length on the binding efficiency and accuracy of GFP expression for a more robust correlation of fluorescence to observed structural changes.

Towards optimization of a fluorescence-based <i>in vivo</i> RNA structural probing assay

247th National Meeting (2014)

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Towards optimization of a fluorescence-based in vivo RNA structural probing assay