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A Fluorescent Split Aptamer for Visualizing RNA–RNA Assembly In Vivo
journal contribution
posted on 2017-05-26, 12:21 authored by Khalid
K. Alam, Kwaku D. Tawiah, Matthew F. Lichte, David Porciani, Donald H. BurkeRNA–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 in vivo. 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 in vitro and improves when cotranscribed, rather than when assembled from
purified components. Split-Broccoli fluorescence
is digital in vivo and retains functional modularity
when fused to RNAs that regulate circuit function through RNA–RNA
hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer
system to operate in vivo. 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.