Quantification and Real-Time Tracking of RNA in Live Cells Using Sticky-Flares*

Abstract

We report a novel spherical nucleic acid (SNA) gold nanoparticle conjugate, termed the Sticky-flare, which enables facile quantification of RNA expression in live cells and spatiotemporal analysis of RNA transport and localization. The Sticky-flare is capable of entering live cells without the need for transfection agents and recognizing target RNA transcripts in a sequence-specific manner. On recognition, the Sticky-flare transfers a fluorophore-conjugated reporter to the transcript, resulting in a turning on of fluorescence in a quantifiable manner and the fluorescent labeling of targeted transcripts. The latter allows the RNA to be tracked via fluorescence microscopy as it is transported throughout the cell. We use this novel nanoconjugate to analyze the expression level and spatial distribution of β-actin mRNA in HeLa cells and to observe the real-time transport of β-actin mRNA in mouse embryonic fibroblasts. Furthermore, we investigate the application of Stickyflares for tracking transcripts that undergo more extensive compartmentalization by fluorophore-labeling U1 small nuclear RNA and observing its distribution in the nucleus of live cells.

Neuronal mitochondria transport Pink1 mRNA via Synaptojanin 2 to support local mitophagy

“…Likewise, the beta actin mRNA has been found to localize to mitochondria in HeLa cells (Briley et al, 2015), supporting the physiological relevance of a closer association between mitochondria and this transcript.…”

Molecular spherical nucleic acids

“…Importantly, despite being highly negatively charged, SNAs rapidly enter cells [via scavenger receptor-mediated endocytosis in the case of endothelial cells (19)], making it possible to deliver large amounts of nucleic acids and other payloads into cells without the need for cocarriers (11,12,20,21). These properties have made SNAs useful as probes for in vitro DNA and RNA detection (22)(23)(24) and lead compounds for gene regulation (25,26), chemotherapy (11,12,20,27), and immune system modulation (28). In addition, SNAs are the central building blocks for crystal engineering strategies based upon programmable assembly concepts (29,30).…”

Circulating tumor cell profiling for precision oncology

“…Fluorescence in situ hybridization (FISH) is a cytogenetic technique that uses fluorescent probes to specifically detect the presence of specific DNA or RNA sequences in individual cells [83]. FISH might require labeling with multiple fluorescent probes to attain a sufficient signal, which might preclude accurate quantification.…”