Redesigning Peppers RNA Imaging System: an Investigation of In Vivo and In Vitro Performance

Mendik, Caitlyn

Undergraduate Honors Thesis

Redesigning Peppers RNA Imaging System: an Investigation of In Vivo and In Vitro Performance Público Deposited

Analytics

Citations

Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/ms35tb04h

Abstract

RNA is a central component of biochemistry as it carries genetic information, has enzymatic activity, mediates protein biodiversity, and supports other cellular functions. Scientists are continually developing new tools for studying RNA and applying it to fields of research such as medicine and molecular biology. RNA imaging provides one avenue for characterizing RNA by looking at trafficking, localization, and lifetime in living cells. Imaging tools have been on the rise in the past 20 years, providing innovative methods for looking at this nucleic acid under a microscope. Peppers is one of these tools designed by Chen et al. at the East China University of Science and Technology. Peppers is an RNA aptamer engineered to bind a fluorescent probe in a stem loop. The 8Peppers aptamer is the optimized form of their probe and is not repeating units of one aptamer, but a long hairpin turn with eight binding spots for probe; this 8Peppers aptamer has been shown to have difficulties folding into native conformation. This paper aims to redesign Peppers RNA imaging system to produce an effective imaging system with enhanced folding capabilities. This paper looks at its in vitro capabilities by fluorescence assay of RNA binding to the probe, HBC620. The redesigned Peppers imaging system is further tested by live cell imaging to determine the efficacy of redesigning the structure, stem, and synonymization of non-consequential regions. The experiments did not show an enhanced fluorescence turn-on or folding capabilities of redesigned Peppers compared to Chen’s original Peppers imaging system. However, the original Peppers imaging tool did not have reproducible results and illustrated the need for a comprehensive and standardized characterization system for RNA imaging tools to be dependable.

Creator