Dual Spatially Localized DNA Walker for Fast and Efficient RNA Detection.

DNA walkers, which are synthetic nanodevices that drive the processive movement of nucleic acids along a well-designed track, have emerged as a powerful tool in biosynthesis, biocomputing, and biosensing due to their exquisite programmability, good biocompatibility, and efficient signal amplification capacity. However, many existing approaches are still hindered by limited reaction kinetics. Herein, we designed a dual spatially localized DNA walker that utilized bipedal catalysts to drive high-speed stochastic movement along three-dimensional tracks via a proximity-driven catalytic hairpin assembly. We demonstrated that the dual colocalization of autocatalytic circuits significantly increased their local concentrations and accelerated reaction kinetics through proximity. We also showed that the use of bipedal catalysts further improved reaction rates compared with unipedal catalysts. Taking advantage of these unique features, we constructed an RNA-responsive PCHA walker for mRNA imaging in live cells, providing a novel and efficient tool for biomolecule detection and biological functions regulation.

Genetically encodable tagging and sensing systems for fluorescent RNA imaging.

Live cell imaging of RNAs is crucial to interrogate their fundamental roles in various biological processes. The highly spatiotemporal dynamic nature of RNA abundance and localization has presented great challenges for RNA imaging. Genetically encodable tagging and sensing (GETS) systems that can be continuously produced in living systems have afforded promising tools for imaging and sensing RNA dynamics in live cells. Here we review the recent advances of GETS systems that have been developed for RNA tagging and sensing in live cells. We first describe the various GETS systems using MS2-bacteriophage-MS2 coat protein, pumilio homology domain and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9/13 for RNA labeling and tracking. The progresses of GETS systems for fluorogenic labeling and/or sensing RNAs by engineering light-up RNA aptamers, CRISPR-Cas9 systems and RNA aptamer stabilized fluorogenic proteins are then elaborated. The challenges and future perspectives in this field are finally discussed. With the continuing development, GETS systems will afford powerful tools to elucidate RNA biology in living systems.