A Fluorescent Split Aptamer for Visualizing RNA–RNA Assembly <i>In Vivo</i>

Published on 2018-02-10T00:04:05Z (GMT) by
RNA–RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA–RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed <i>in vivo</i>. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second-order kinetics <i>in vitro</i> and improves when cotranscribed, rather than when assembled from purified components. <i>Split-Broccoli</i> fluorescence is digital <i>in vivo</i> and retains functional modularity when fused to RNAs that regulate circuit function through RNA–RNA hybridization, as demonstrated with an RNA Toehold switch. <i>Split-Broccoli</i> represents the first functional split-aptamer system to operate <i>in vivo</i>. It offers a genetically encoded and nondestructive platform to monitor and exploit RNA–RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA–RNA hybrids.

Cite this collection

Alam, Khalid

K.; Tawiah, Kwaku D.; Lichte, Matthew F.; Porciani, David; H. Burke, Donald (2018): A Fluorescent Split Aptamer for Visualizing RNA–RNA

Assembly In Vivo. ACS Publications. Collection.