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"DEVELOPING A GENETICALLY ENCODABLE PROBE SYSTEM FOR IMAGING RNA IN LIVE CELLS"

by
Wei Yang
Microbiology and Molecular Genetics Program
B.S. 2007, Shenyang Pharmaceutical University, China



Thesis Advisor: Sanjay Tyagi, Ph.D.
Associate Professor
Public Health Research Institute

Tuesday, May 22, 2012
2:00 P.M., 2:00 P.M.


Abstract

Imaging the distribution and the dynamics of mRNA in live cells can provide powerful insights into diverse biological phenomena. We have developed genetically encodable probes to image RNAs in live cells. This system is composed of an RNA probe that can be expressed inside the cell and report the presence of a target RNA, by assuming a specific structure (aptamer), which binds to a dye and renders it fluorescent. The probe is composed of three elements: I) an aptamer, which when folded into its functional conformation, is able to bind and elicit fluorescence in the dye; II) a probe element, which is complementary to the target RNA; III) an anti-aptamer element, which is complementary to a region of the aptamer sequence. When the target RNA is absent, the functional conformation of the aptamer is abrogated due to its hybridization to the anti-aptamer element. However, when the target RNA is present, the probe element binds to the target RNA to form a rigid hybrid that separates the anti-aptamer from the aptamer. Thus, the aptamer resumes its functional conformation and elicits fluorescence from the dye. We have successfully designed and tested probes in vitro based on a recently discovered aptamer called Spinach, and derived rules for optimizing the probe performance from more than 60 probe designs. In parallel to these efforts aimed at probe design, we synthesized and characterized a set of ten new compounds that can fluoresce in colors ranging from deep blue to far red that will be useful for multiplexing the RNA imaging. Using SELEX, we isolated two RNA aptamers that bind to and elicit fluorescence in one of the compounds. As the level of fluorescence generated from affinity-isolated aptamers was moderate, we developed a new method for the isolation of RNA aptamers for their ability to elicit fluorescence in dyes. These genetically encodable probes will aid in exploration of RNA dynamics in many different biological contexts.


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