Development of new tags for RNA visualization

PROJECT SUMMARY RNA plays numerous roles in biology. In addition to its canonical role in the central dogma as an intermediary between DNA genomes and protein products of translation, a myriad of roles have been described for this molecule, including gene silencing (siRNA), regulation of translation (bacterial riboswitches), and the translation machinery itself (rRNA). Despite the importance of these roles in biology, tools for imaging RNA are not as robust as the suite of fluorescent proteins that exist for readout of protein machinery. This project seeks to address this need by developing new fluorescent aptamer tags for RNA visualization. This strategy has enjoyed considerable success, particularly in bacterial systems, but deployment in complex systems (eukaryotic, whole- organism) has been challenging. Recent work on RNA folding has shown that a noncanonical RNA structure (a G Quadruplex) may impair the use of aptamers as tags in these systems. This project seeks to select fluorescent aptamers that are not G-Quadruplexes. We will seek select non-G-quadruplex-forming aptamers against a range of azo dyes (Aim 1) and endogenous pigments (Aim 2) for robust live-cell imaging, and a long-range goal of whole-organism RNA imaging. We will use these tags to characterize Lin28-mRNA interactions, which is not possible with existing G-quadruplex-forming aptamers, because Lin28 is known to unfold G-quadruplexes in RNA. We will also develop benchmarking tags for our selected aptamers by selecting non-G- quadruplex-forming aptamers to known aptamer ligands, which we will use to compare our selected aptamers.