Live-cell RNA Imaging Research Articles

Decoding Arc transcription: a live-cell study of stimulation patterns and transcriptional output.

Activity-regulated cytoskeleton-associated protein (Arc) plays a crucial role in synaptic plasticity, a process integral to learning and memory. Arc transcription is induced within a few minutes of stimulation, making it a useful marker for neuronal activity. However, the specific neuronal activity patterns that initiate Arc transcription have remained elusive due to the inability to observe mRNA transcription in live cells in real time. Using a genetically encoded RNA indicator (GERI) mouse model that expresses endogenous Arc mRNA tagged with multiple GFPs, we investigated Arc transcriptional activity in response to various electrical field stimulation patterns. The GERI mouse model was generated by crossing the Arc-PBS knock-in mouse, engineered with binding sites in the 3' untranslated region (UTR) of Arc mRNA, and the transgenic mouse expressing the cognate binding protein fused to GFP. In dissociated hippocampal neurons, we found that the pattern of stimulation significantly affects Arc transcription. Specifically, theta-burst stimulation consisting of high-frequency (100 Hz) bursts delivered at 10 Hz frequency induced the highest rate of Arc transcription. Concurrently, the amplitudes of nuclear calcium transients also reached their peak with 10 Hz burst stimulation, indicating a correlation between calcium concentration and transcription. However, our dual-color single-cell imaging revealed that there were no significant differences in calcium amplitudes between Arc-positive and Arc-negative neurons upon 10 Hz burst stimulation, suggesting the involvement of other factors in the induction of Arc transcription. Our live-cell RNA imaging provides a deeper insight into the complex regulation of transcription by activity patterns and calcium signaling pathways.

Single-molecule live-cell RNA imaging with CRISPR-Csm.

High-resolution, real-time imaging of RNA is essential for understanding the diverse, dynamic behaviors of individual RNA molecules in single cells. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for efficient, direct visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we tracked individual endogenous NOTCH2 and MAP1B mRNA transcripts in living cells and identified two distinct localization mechanisms: co-translational translocation of NOTCH2 mRNA at the endoplasmic reticulum, and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.

Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA.

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

An Orthogonal CRISPR/dCas12a System for RNA Imaging in Live Cells.

CRISPR/Cas technology has made great progress in the field of live-cell imaging beyond genome editing. However, effective and easy-to-use CRISPR systems for labeling multiple RNAs of interest are still needed. Here, we engineered a CRISPR/dCas12a system that enables the specific recognition of the target RNA under the guidance of a PAM-presenting oligonucleotide (PAMmer) to mimic the PAM recognition mechanism for DNA substrates. We demonstrated the feasibility and specificity of this system for specifically visualizing endogenous mRNA. By leveraging dCas12a-mediated precursor CRISPR RNA (pre-crRNA) processing and the orthogonality of dCas12a from different bacteria, we further demonstrated the proposed system as a simple and versatile molecular toolkit for multiplexed imaging of different types of RNA transcripts in live cells with high specificity. This programmable dCas12a system not only broadens the RNA imaging toolbox but also facilitates diverse applications for RNA manipulation.

Fine-tuning of highly bright benzo[c,d]indole-oxazolopyridine cyanine dye for nucleolar RNA imaging in living cells

Here we report on fine-tuning of highly bright fluorogenic cyanine dye, benzo[c,d]indole-oxazolo[5,4-c]pyridine (BIOP: λem = 570 nm, Φfree = 0.00038, Φbound = 0.52), recently developed by our group for nucleolar RNA imaging in living cells. We tuned an emission maximum to the longer wavelength by replacing oxazolopyridine unit with its isomer. The resulting probe with oxazolo[4,5-b]pyridine, named BIOP [4,5-b], exhibited a significant off-on signaling ability for RNA (λem = 580 nm, Φfree = 0.002, Φbound = 0.46) that almost compared with BIOP. BIOP [4,5-b] was applicable to live-cell imaging, where wash-free protocol was available. Importantly, the slight change in spectral features would be expected to minimize the false signal from BIOP [4,5-b] in co-staining experiments with typical green-emissive dyes. In addition, thanks to the unique spectral shape that is typical of cyanine dyes, we demonstrate that yellow-emissive BIOP [4,5-b] also did work as a pseudo red-emissive dye (λem > 600 nm) for live-cell RNA imaging, for which we just switch filter set to typical one for real red-emissive dyes (Ex 560/40 nm; Em 645/75 nm).

Recent advances in methods for live-cell RNA imaging.

As one of the most fundamental building blocks of life, RNA plays critical roles in diverse biological processes, from X chromosome inactivation, genome stability maintenance, to embryo development. Being able to visualize the localization and dynamics of RNA can provide critical insights into these fundamental processes. In this review, we provide an overview of current methods for live-cell RNA imaging with a focus on methods for visualizing RNA in living mammalian cells with single-molecule resolution.

Fast-exchanging spirocyclic rhodamine probes for aptamer-based super-resolution RNA imaging

Live-cell RNA imaging with high spatial and temporal resolution remains a major challenge. Here we report the development of RhoBAST:SpyRho, a fluorescent light-up aptamer (FLAP) system ideally suited for visualizing RNAs in live or fixed cells with various advanced fluorescence microscopy modalities. Overcoming problems associated with low cell permeability, brightness, fluorogenicity, and signal-to-background ratio of previous fluorophores, we design a novel probe, SpyRho (Spirocyclic Rhodamine), which tightly binds to the RhoBAST aptamer. High brightness and fluorogenicity is achieved by shifting the equilibrium between spirolactam and quinoid. With its high affinity and fast ligand exchange, RhoBAST:SpyRho is a superb system for both super-resolution SMLM and STED imaging. Its excellent performance in SMLM and the first reported super-resolved STED images of specifically labeled RNA in live mammalian cells represent significant advances over other FLAPs. The versatility of RhoBAST:SpyRho is further demonstrated by imaging endogenous chromosomal loci and proteins.

Sequence-Specific Fluorescence Turn-On Sensing of RNA by DNA Probes Incorporating the Tricyclic Cytidine Analogue DEAtC.

Sequence-specific fluorescent probes for RNA are widely used in microscopy applications such as fluorescence in situ hybridization and a growing number of newer approaches to live-cell RNA imaging. The sequence specificity of most of these approaches relies on differential hybridization of the probe to the correct target. Competing sequences with only one or two base mismatches are prone to causing off-target recognition. Here, we report the sequence-specific fluorescent detection of model RNA targets using a tricyclic cytidine analogue DEAtC that is included as a surrogate for natural cytidine in DNA probe strands and that reports directly on Watson-Crick base pairing. The DEAtC-containing DNA oligonucleotide probes exhibit an average 8-fold increase in fluorescence intensity when hybridized to matched RNA with DEAtC base paired with G and little fluorescence turn-on when DEAtC is base paired with A. Duplex structure determination by NMR, time-resolved fluorescence studies, and Stern-Volmer quenching experiments suggest that the combination of greater π stacking and narrower grooves in the A-form DNA-RNA heteroduplex provides additional shielding and favorable electronic interactions between bases, explaining why DEAtC's fluorescence turn-on response to RNA targets is typically 3-fold greater than for DNA targets.

Enhanced single RNA imaging reveals dynamic gene expression in live animals.

Imaging endogenous mRNAs in live animals is technically challenging. Here we describe an MS2-based signal amplification with the Suntag system that enables live-cell RNA imaging of high temporal resolution and with 8xMS2 stem-loops, which overcomes the obstacle of inserting a 1,300 nt 24xMS2 into the genome for the imaging of endogenous mRNAs. Using this tool, we were able to image the activation of gene expression and the dynamics of endogenous mRNAs in the epidermis of live C. elegans.

Precise transcript targeting by CRISPR-Csm complexes

Robust and precise transcript targeting in mammalian cells remains a difficult challenge using existing approaches due to inefficiency, imprecision and subcellular compartmentalization. Here we show that the clustered regularly interspaced short palindromic repeats (CRISPR)-Csm complex, a multiprotein effector from type III CRISPR immune systems in prokaryotes, provides surgical RNA ablation of both nuclear and cytoplasmic transcripts. As part of the most widely occurring CRISPR adaptive immune pathway, CRISPR-Csm uses a programmable RNA-guided mechanism to find and degrade target RNA molecules without inducing indiscriminate trans-cleavage of cellular RNAs, giving it an important advantage over the CRISPR-Cas13 family of enzymes. Using single-vector delivery of the Streptococcus thermophilus Csm complex, we observe high-efficiency RNA knockdown (90–99%) and minimal off-target effects in human cells, outperforming existing technologies including short hairpin RNA- and Cas13-mediated knockdown. We also find that catalytically inactivated Csm achieves specific and durable RNA binding, a property we harness for live-cell RNA imaging. These results establish the feasibility and efficacy of multiprotein CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

Live-Cell RNA Imaging with Metabolically Incorporated Fluorescent Nucleosides.

Fluorescence imaging is a powerful method for probing macromolecular dynamics in biological systems; however, approaches for cellular RNA imaging are limited to the investigation of individual RNA constructs or bulk RNA labeling methods compatible primarily with fixed samples. Here, we develop a platform for fluorescence imaging of bulk RNA dynamics in living cells. We show that fluorescent bicyclic and tricyclic cytidine analogues can be metabolically incorporated into cellular RNA by overexpression of uridine-cytidine kinase 2. In particular, metabolic feeding with the tricyclic cytidine-derived nucleoside tC combined with confocal imaging enables the investigation of RNA synthesis, degradation, and trafficking at single-cell resolution. We apply our imaging modality to study RNA metabolism and localization during the oxidative stress response and find that bulk RNA turnover is greatly accelerated upon NaAsO2 treatment. Furthermore, we identify cytoplasmic RNA granules containing RNA transcripts generated during oxidative stress that are distinct from canonical stress granules and P-bodies and co-localize with the RNA helicase DDX6. Taken together, our work provides a powerful approach for live-cell RNA imaging and reveals how cells reshape RNA transcriptome dynamics in response to oxidative stress.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Research progress of live-cell RNA imaging techniques

RNA molecules play diverse roles in many physiological and pathological processes as they interact with various nucleic acids and proteins. The various biological processes of RNA are highly dynamic. Tracking RNA dynamics in living cells is crucial for a better understanding of the spatiotemporal control of gene expression and the regulatory roles of RNA. Genetically encoded RNA-tagging systems include MS2/MCP, PP7/PCP, boxB/λN22 and CRISPR-Cas. The MS2/MCP system is the most widely applied, and it has the advantages of stable binding and high signal-to-noise ratio, while the realization of RNA imaging requires gene editing of the target RNA, which may change the characteristics of the target RNA. Recently developed CRISPR-dCas13 system does not require RNA modification, but the uncertainty in CRISPR RNA (crRNA) efficiency and low signal-to-noise ratio are its limitations. Fluorescent dye-based RNA-tagging systems include molecular beacons and fluorophore-binding aptamers. The molecular beacons have high specificity and high signal-to-noise ratio; Mango and Peppers outperform the other RNA-tagging system in signal-to-noise, but they also need gene editing. Live-cell RNA imaging allows us to visualize critical steps of RNA activities, including transcription, splicing, transport, translation (for message RNA only) and subcellular localization. It will contribute to studying biological processes such as cell differentiation and the transcriptional regulation mechanism when cells adapt to the external environment, and it improves our understanding of the pathogenic mechanism of various diseases caused by abnormal RNA behavior and helps to find potential therapeutic targets. This review provides an overview of current progress of live-cell RNA imaging techniques and highlights their major strengths and limitations.

Rational Design of Self-Quenched Rhodamine Dimers as Fluorogenic Aptamer Probes for Live-Cell RNA Imaging

With the growing interest in the understanding of the importance of RNAs in health and disease, detection of RNAs in living cells is of high importance. Fluorogenic dyes that light up specifically selected RNA aptamers constitute an attractive direction in the design of RNA imaging probes. In this work, based on our recently proposed concept of a fluorogenic dimer, we aim to develop a robust molecular tool for intracellular RNA imaging. We rationally designed a fluorogenic self-quenched dimer (orange Gemini, o-Gemini) based on rhodamine and evaluated its capacity to light up its cognate aptamer o-Coral in solution and live cells. We found that the removal of biotin from the dimer slightly improved the fluorogenic response without losing the affinity to the cognate aptamer (o-Coral). On the other hand, replacing sulforhodamine with a carboxyrhodamine produced drastic improvement of the affinity and the turn-on response to o-Coral and, thus, a better limit of detection. In live cells expressing o-Coral-tagged RNAs, the carboxyrhodamine analogue of o-Gemini without a biotin unit displayed a higher signal as well as faster internalization into the cells. We suppose that less hydrophilic carboxyrhodamine compared to sulforhodamine can more readily penetrate through the cell plasma membrane and, together with its higher affinity to o-Coral, provide the observed improvement in the imaging experiments. The promiscuity of the o-Coral RNA aptamer to the fluorogenic dimer allowed us to tune a fluorogen chemical structure and thus drastically improve the fluorescence response of the probe to o-Coral-tagged RNAs.

DNA-Templated Bioorthogonal Reactions via Catalytic Hairpin Assembly for Precise RNA Imaging in Live Cells.

There has been a significant interest in developing proximity-induced bioorthogonal reactions for nucleic acid detection and imaging, owing to their high specificity and tunable reaction kinetics. Herein, we reported the first design of a fluorogenic sensor by coupling a bioorthogonal reaction with a DNA cascade circuit for precise RNA imaging in live cells. Two DNA hairpin probes bearing tetrazines or vinyl ether caged fluorophores were designed and synthesized. Upon target mRNA triggering catalytic hairpin assembly, the chemical reaction partners were brought in a spatial proximity to yield high effective concentrations, which dramatically facilitated the bioorthogonal reaction efficiency to unmask the vinyl ether group to activate fluorescence. The proposed fluorogenic sensor was demonstrated to have a high signal-to-noise ratio up to ∼30 fold and enabled the sensitive detection of target mRNA with a detection limit of 4.6 pM. Importantly, the fluorogenic sensor presented low background signals in biological environments due to the unique "click to release" feature, avoiding false positive results caused by unspecific degradation. We also showed that the fluorogenic sensor could accurately image mRNA in live cells and distinguish the relative mRNA expression levels in both tumor and normal cells. Benefiting from these significant advantages, our method provides a useful tool for basic studies of bioorthogonal chemistry and early clinical diagnosis.

Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers.

The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based on the CRISPR-dPspCas13b system with fluorescent RNA aptamers in sgRNA (CasFAS) in live cells. Using modified sgRNA attached to fluorescent RNA aptamers that showed reduced background fluorescence, this approach provides a simple, sensitive way to image and track endogenous RNA with high accuracy and efficiency. In addition, color switching can be easily achieved by changing the fluorogenic dye analogues in living cells through user-friendly washing and restaining operations. CasFAS is compatible with orthogonal fluorescent aptamers, such as Broccoli and Pepper, enabling multiple colors RNA labeling or intracellular RNA-RNA interaction imaging. Finally, the visualization of severe fever with thrombocytopenia syndrome virus (SFTSV) was achieved by CasFAS, which may facilitate further studies on this virus.

The lupus autoantigen La/Ssb is an Xist-binding protein involved in Xist folding and cloud formation.

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

Genetically Encoded Sensor Enables Endogenous RNA Imaging with Conformation-Switching Induced Fluorogenic Proteins.

Genetically encoded molecular tools are crucial for live cell RNA imaging, and few are available for endogenous RNA imaging. We develop a new genetically encoded sensor using conformation switching RNA induced fluorogenic proteins that enable multicolor and signal-amplified imaging of endogenous RNAs. The sensor system is designed with an RNA sensing module and a degron-fused fluorescent protein reporter. Target RNA induces conformation switching of the RNA sensing module to form RNA aptamers that stabilize the degron-fused protein for fluorogenic imaging. This sensor is demonstrated for high-contrast imaging of survivin mRNA abundance and dynamics in live cells. Moreover, the sensor system is extended to a multicolor palette by screening fluorogenic proteins of distinct colors, and engineered into a signal amplifier using the split fluorescent protein design. The sensor is further exploited for imaging lncRNA MALAT-1 and its translocation dynamics during mitosis. Our sensor system can afford a valuable platform for RNA imaging in biomedical research and clinical theranostics.

The Lupus Autoantigen La/Ssb is an <i>Xist</i>-Binding Protein Involved in <i>Xist</i> Folding and Cloud Formation

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain and/or the ATP binding motif play critical roles in XCI. In mutant cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.

Visualization of cell-type dependent effects of anti-E2 antibody and interferon-gamma treatments on localization and expression of Broccoli aptamer-tagged alphavirus RNAs

Sindbis virus (SINV) is an alphavirus that causes age-dependent encephalomyelitis in mice. Within 7–8 days after infection infectious virus is cleared from neurons through the antiviral effects of antibody and interferon-gamma (IFNγ), but RNA persists. To better understand changes in viral RNA associated with immune-mediated clearance we developed recombinant strains of SINV that have genomic and subgenomic viral RNAs tagged with the Broccoli RNA aptamer that binds and activates a conditional fluorophore for live cell imaging of RNA. Treatment of SINV-Broccoli-infected cells with antibody to the SINV E2 glycoprotein had cell type-specific effects. In BHK cells, antibody increased levels of intracellular viral RNA and changed the primary location of genomic RNA from the perinuclear region to the plasma membrane without improving cell viability. In undifferentiated and differentiated AP7 (dAP7) neuronal cells, antibody treatment decreased levels of viral RNA. Occasional dAP7 cells escaped antibody-mediated clearance by not expressing cell surface E2 or binding antibody to the plasma membrane. IFNγ decreased viral RNA levels only in dAP7 cells and synergized with antibody for RNA clearance and improved cell survival. Therefore, analysis of aptamer-tagged SINV RNAs identified cell type- and neuronal maturation-dependent responses to immune mediators of virus clearance.