Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
A New Approach to Model and Directly Control "Co-transcriptional" RNA Folding
RNAs are synthesized exclusively in a 5' to 3' direction and it is well established that many (particularly long noncoding, but also riboswitch) RNAs begin to fold before the transcript is fully synthesized. Indeed, in many cases, directional folding is thought to limit the formation of nonproductive, nonnative structures that might arise from a more random condensation of secondary and tertiary structures or to facilitate formation of necessary (and transient on pathway) folding intermediates. Site-specifically programmed pauses in transcription are often necessary to direct these interactions.
As with co-translational protein folding, the tools to study such RNA folding simply do not exist. We would like to be able to pause transcription to test the role of sequence-encoded pausing, or to interrogate or perturb potential folding intermediates, and then chase the RNA to the final folded product. We would like to be able to do this without changing the sequence of the folded RNA.
Using tools from synthetic biology and nucleic acid nanotechnology (specifically, toehold mediated strand displacement), we have demonstrated and are further developing an alternative approach to direct and control 5' to 3' sequential RNA folding. The approach allows the introduction of pauses into the sequential release and allows the experimenter to halt and then resume RNA release/folding. Initial studies use the malachite green binding RNA aptamer, with a simple fluorescence readout of formation of the final structure. Subsequent studies will footprint intermediates with a variety of probes to follow the progress of folding and detect intermediates.
Aruni Karunanayake Mudiyanselage
Department of Chemistry