DNA-Sequence-Encoded Rolling Circle Amplicon for Single-Cell RNA Imaging

Abstract

The expression and spatial profile of multiple RNA species at high precision in single cells is key information for understanding cellular behaviors and functions. Fluorescence microscopy is a powerful tool, but its multiplexing ability is limited by the number of spectrally distinct fluorophores. Here, a DNA-sequence-encoded fluorescence barcoding method based on thermodynamically tuning DNA hybridization was applied for fluorescence tagging RNAs, which allows for highly multiplexed imaging of RNA in single cells with single-molecule and singlenucleotide resolution.

Protein‐Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer‐Containing DNA Primer

“…With regard to POC tests, RCA has several advantages over other ITA methods. It can be performed at room temperature, requires minimal reagents (a linear and circular primer and one processing enzyme) and is compatible with many types of assay outputs . Our groups have reported on several methods to modulate RCA using a protein target, including: integration of a protein‐binding aptamer into a circular template to modulate RCA; binding of a protein to release an aptamer‐primer conjugate from reduced graphene oxide (rGO) to initiate RCA; using an aptamer to inhibit a linear primer from being extended along a circular template until target binds to release the aptamer; and use of protein‐binding DNAzymes that can release a DNA primer upon catalytic cleavage of an embedded ribonucleic acid to initiate RCA .…”

Immuno-SABER enables highly multiplexed and amplified protein imaging in tissues

“…RCA offers high levels of amplification and potential for multiplexing, but in situ enzymatic reaction is hard to control or tune for individual targets 18 . Both TSA and RCA may also lead to blurring of signals and decreased resolution, respectively due to spreading of the tyramide molecules or the large size of the amplicons (reaching from 250 nm to over ~1 μm radius 19 , 20 ). Branched DNA assemblies 21 , 22 , such as RNAscope 23 , generate complex tree structures for stable binding of fluorescent DNA strands, whereas Hybridization Chain Reaction (HCR) utilizes the triggered assembly of metastable fluorophore-conjugated hairpins 24 – 26 .…”

In Situ Amplification‐Based Imaging of RNA in Living Cells

“…[40] In RT-PCR, atarget RNAofinterest is first reverse transcribed into complementary DNA(cDNA) transcripts by reverse transcriptase.T hen, the newly-produced cDNAi s amplified through traditional PCR in the presence of thermostable polymerase,r esulting in indirect amplification-based detection of the target RNA. [44][45][46][47][48][49][50][51][52][53] In FISH, enzymes that can catalyze the amplification of nucleic acid under ambient temperature are generally transferred into cells,t hen the target nucleic acid of interest is amplified, leading to af luorescence signal. [41][42][43] In addition, isothermal enzymatic signal amplificationbased fluorescence in situ hybridization (FISH) assay has been developed for single-cell RNAi maging.…”