In Situ Spatial Complementation of Aptamer‐Mediated Recognition Enables Live‐Cell Imaging of Native RNA Transcripts in Real Time

Abstract

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single-cell resolution. In contrast to routine fluorescent-protein-based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer-initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)-mimicking turn-on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split-Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore-binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high-contrast and real-time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single-cell visualization and mechanistic analysis of macromolecular interactions.

RNA Structure and Cellular Applications of Fluorescent Light‐Up Aptamers

“…This miR-21 sensor was genetically encoded and used to quantitatively detect miR-21 in different live mammalian (tumor) cells.T o detect endogenous mRNAi nm ammalian cells,W ang et al very recently developed as plit-Broccoli system. [91,92] Here, the endogenous mRNAs erves as at emplate to reconstitute Broccoli from two halves that adjacently bind to the mRNA of interest ( Figure 9D). This approach leads to lower background fluorescence and sufficient brightness for real-time imaging of b-actin mRNAd istribution in live HeLa cells.…”

“…Am odified Spinach FLAP was engineered to offer ar everse complementary binding site for miRNAm iR-122 by Meyers and co-workers in 2017. [91,92] Here, the endogenous mRNAs erves as at emplate to reconstitute Broccoli from two halves that adjacently bind to the mRNA of interest ( Figure 9D). Sufficient brightness for measurements in live HEK293T cells, however, were only possible with asix-tandem repeat.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…Wang et al developed a “split‐broccoli” system for imaging mRNA in live cells . The Broccoli aptamer was split into two fragments and genetically encoded into live mammalian cells.…”

PITTCON 2019 Award Winners

“…Fan's current research is based on the development of nucleic acid tools for biomolecular sensing and live‐cell imaging. In Communications in Angewandte Chemie he has reported on live‐cell imaging of native RNA transcripts and near‐infrared imaging of colorectal cancers using activatable nanoprobes . Fan is a member of the Editorial Advisory Boards of ChemBioChem and ChemNanoMat .…”