Rather than tiling probes along a transcript, hybridization chain reaction (HCR)-FISH 8 and signal amplification by exchange reaction (SABER)-FISH 9 amplify primary probes with hairpin probes and concatemers, respectively, to tile fluorescent secondary probes along a primary probe. Similarly, click-amplifying FISH (clampFISH) iteratively hybridizes padlock probes to target mRNAs and ligates them using bio-orthogonal click chemistry before hybridization with a FISH probe, resulting in a >400-fold signal amplification per single molecule of RNA 7. RNAscope leverages multiple tiled primary, secondary and tertiary DNA oligonucleotide probes 6. Innovation of single-molecule RNA imaging continues to build on smFISH and RCA-FISH to further improve detection efficiency, increase brightness and reduce overall cost. Rather than tiling multiple probes to a desired mRNA target, rolling-circle amplification (RCA)-FISH first hybridizes and ligates a padlock probe specific to the mRNA target and then amplifies the padlock probe using RCA 4, 5. In 2008, the method was further refined to detect mRNAs at single-molecule resolution by probing each mRNA with 48 DNA probes, each labelled with single fluorochromes 3. In 1998, single-molecule FISH (smFISH) was developed using a complementary DNA (cDNA) oligonucleotide synthesized with five fluorochromes per probe 2. In 1982, Singer and Ward were among the first to demonstrate fluorescence in situ hybridization (FISH) for RNA detection by probing actin messenger RNA (mRNA) with rhodamine-conjugated avidin binding to a DNA probe with incorporated biotinylated 2′-deoxyuridine-5′-triphosphate (dUTP) 1. ‘ N hybridizations’ indicates N rounds of hybridizations Rehyb., rehybridization. Recently, conjugates of fluorescent protein and RNA-binding Cas proteins were developed to visualize endogenous RNA in living cells. Microinjected fluorescent dUTPs can be incorporated into endogenous RNA, enabling visualization. Fluorogenic RNA takes advantage of an RNA aptamer that can encase the fluorophore structure 4-hydroxybenzylidene imidazolinone and emit light. The molecular beacon, which remains dark until hybridized to a target RNA, can be delivered to cells to image endogenous RNA. The stem-loop system takes advantage of a fluorescent protein attached to a viral coat protein that can bind to an RNA stem loop, such as MS2, enabling single-molecule resolution for the first time. b, The evolution of live-cell RNA imaging started with the microinjection of fluorescent RNA in 1997. Combinatorial FISH methods (MERFISH, seqFISH) and in situ sequencing (ISS, FISSEQ, STARmap, HybISS, ExSeq, Seq-Scope) methods enable multiplexing. Fluorescently labelled DNA probes bind to these amplified sites and emit much brighter signals compared to smFISH. RCA-FISH, clampFISH, HCR-FISH, RNAscope and SABER-FISH enhance signals by amplifying the primary probes that hybridize to the RNA target by RCA, by secondary and tertiary probes, or by primer-exchange reaction (PER). smFISH amplifies signals by utilizing multiple fluorescent DNA probes binding to a single RNA target. In this Review we discuss the developments in RNA imaging and the RNA biology they have and are poised to unravel.Ī, The development of fixed-cell RNA imaging from the development of fluorescent RNA detection in 1982. These RNA imaging tools take advantage of recent and rapid innovation in fluorescent microscopy, image processing, DNA chemistry and next-generation sequencing to achieve multiple milestones, including single-molecule sensitivity, super-resolution, multiplexing and live-cell RNA tracking.

Towards this goal, in the past four decades groups have developed and advanced RNA imaging tools for both fixed and live cells (Fig. To delve deeper into the when and where, methods to visualize RNA within cells are required. Traditional biochemistry and genetic tools have elucidated some of the what and the how, such as the identities and functions of proteins and non-coding RNAs (ncRNAs) involved in each step of RNA processing. These steps are highly coordinated and tightly regulated in both spatial and temporal domains. After transcription, RNA molecules proceed to splicing, localization, translation and degradation. The transformation from DNA to protein is a complex, multi-stage process that revolves around RNA metabolism.

Messenger rna image