Sensitive Imaging of Cellular RNA via Cascaded Proximity-Induced Fluorogenic Reactions

Abstract

Owing to its important biological functions, RNA has become a promising molecular biomarker of various diseases. With a dynamic change in its expression level and a relatively low amount within the complicated biological matrix, signal amplification detection based on DNA probes has been put forward, which is helpful for early diagnosis and prognostic prediction. However, conventional methods are confined to cell lysates or dead cells and are not only time-consuming in sample preparation but also inaccessible to the spatial–temporal information of target RNAs. To achieve live-cell imaging of specific RNAs, both the detection sensitivity and intracellular delivery issues should be addressed. Herein, a new cascaded fluorogenic system based on the combination of hybridization chain reactions (HCRs) and proximity-induced bioorthogonal chemistry is developed, in which a bioorthogonal reaction pair (a tetrazine-quenched dye and its complementary dienophile) is brought into spatial proximity upon target RNA triggering the HCR to turn on and amplify the fluorescence in one step, sensitively indicating the cellular distribution of RNA with minimal false positive results caused by unspecific degradation. Facilitated by a biodegradable carrier based on black phosphorus with high loading capacity and excellent biocompatibility, the resulting imaging platform allows wash-free tracking of target RNAs inside living cells.

Tetrazine bioorthogonal chemistry makes nanotechnology a powerful toolbox for biological applications

“…Chen and coworkers have developed tetrazine bioorthogonal ligation-based nanotechnology for the in situ detection of low-abundance RNA in biological environments. 62 The authors claimed that both sensing sensitivity and cellular delivery problems should be overcome to fulfill in vivo imaging and detection of target RNAs. Such concerns had pushed them to develop novel tetrazine bioorthogonal-based fluorescence "turn-on" platform for RNA detection, in which the bioorthogonal ligation motifs (a fluorescence quenched tetrazine dye and a target complementary dienophile) were brought into spatial proximity upon selected RNA triggering the hybridization chain reaction to turned on and amplified the fluorescence signal simultaneously, sensitively demonstrating the RNA distribution inside living cells (Fig.…”

Quantitative Analysis of Mitochondrial RNA in Living Cells with a Dual‐Color Imaging System

“…cytosol, respectively), black phosphorus nanosheet (BPNS) featured good biocompatibility, high loading capability, [23,24] and biodegradability under physiological conditions to give nontoxic products (phosphate and phosphonate ions), [24][25][26][27][28] was functionalized as robust delivery vehicles. Moreover, the high surface-to-volume ratio and periodic atomic grooves on the BPNS surface provide ideal anchoring sites for nucleic acids [29][30][31][32] and protect them from degradation by endogenous nucleases. As a proof of concept, HDCA toward mtRNA encoding NADH dehydrogenase subunit 6 (ND6) (mtRNA ND6 for short) was integrated into mitochondrion-targeted BPNS, BP-PEI-TPP, to detect mtRNA ND6 .…”