(12b) Exploiting Post-Transcriptional Regulatory Interactions for in Vivo RNA Characterization

“Exploiting Post-transcriptional Regulatory Interactions for in vivo RNA Characterization”

Sowa, S.*; Vazquez-Anderson, J.*; Contreras, L.

McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Blvd., CO400 Austin, Texas 78712-1589   

Microbiology Graduate Program, University of Texas at Austin, 100 E. 24th Street, STOP A6500
Austin, Texas  78712

 *These authors contributed equally to these work

RNA structure has been extensively studied using a variety of in vitro and in silico techniques (e.g. crystallography, NMR, footprinting, comparative analysis, etc.). However, to date, only a few RNA structural characterization techniques have been used within an intracellular environment. In addition, although in vitro and in vivo RNA folding share some basic features, is still unclear how RNA folding in vitro compares to in vivo (Schroeder, 2002). In this study, we exploit molecular interactions that trigger activation of GFP to engineer a controllable riboswitch-inspired system capable of detecting structural perturbations in RNAs that result from sequence mutations. In this reporter system, expression of GFP is tuned by controlling local structural changes in an RBS and a cis-blocking element (cb) containing-loop. We explore different riboswitch conformations in the context of their interactions with the tetrahymena ribozyme Group I Intron (GI). Flow cytometry data indicates the potential of this method to detect differences between RNA variants and RNA conformers.  We will discuss various design features of the system, and design algorithms that we have now established for optimal sensitivity. We will also discuss potential mechanisms that describe specific interactions between the target RNA and the designed RNA responsive elements that leads to detection of structural changes.