Live-cell RNA Imaging Research Articles

Hijacking Cas9 for live-cell RNA imaging

Researchers target fluorescent protein–Cas9 fusions to specific mRNAs for tracking in live cells.

DNA Stains as Surrogate Nucleobases in Fluorogenic Hybridization Probes.

The increasing importance assigned to RNA dynamics in cells and tissues calls for probe molecules that enable fluorescence microscopy imaging in live cells. To achieve this goal, fluorescence dyes are conjugated with oligonucleotides so as to provide strong emission upon hybridization with the target molecule. The impressive 10(3)-fold fluorescence intensification observed when DNA stains such as thiazole orange (TO) interact with double-stranded DNA is intriguing and prompted the exploration of oligonucleotide conjugates. However, nonspecific interactions of DNA stains with polynucleotides tend to increase background, which would affect the contrast achievable in live-cell imaging. This Account describes the development of DNA-stain-labeled hybridization probes that provide high signal-to-background. We focus on our contributions in context with related advances from other laboratories. The emphasis will be on the requirements of RNA imaging in live cells. To reduce background, intercalator dyes such as TO were appended to peptide nucleic acid (PNA), which is less avidly recognized by DNA stains than DNA/RNA. Constraining the TO dye as a nucleobase surrogate in "forced intercalation (FIT) probes" improved the target specificity, presumably by helping to prevent unspecific interactions. The enforcement of TO intercalation between predetermined base pairs upon formation of the probe-target duplex provided for high brightness and enabled match/mismatch selectivity beyond stringency of hybridization. We show examples that highlight the use of PNA FIT probes in the imaging of mRNA, miRNA, and lncRNA in living cells. The "FIT approach" was recently extended to DNA probes. Signal brightness can become limiting when low-abundance targets ought to be visualized over cellular autofluorescence. We discuss strategies that further the brightness of signaling by FIT probes. Multilabeling with identical dyes does not solve the brightness issue. To avoid self-quenching, we combined two different yet spectrally overlapping fluorescent base surrogates. A hybridization-sensitive dye serves as a light collector that transfers energy to a brightly emissive acceptor dye. To improve the brilliance of single-dye probes, the "TO-nucleotide" was accompanied by an adjacent locked nucleic acid (LNA) unit. The LNA-constrained FIT probes are responsive and bright, enabling the tracking of mRNA transport in living tissue. We also show that the color repertoire of FIT probes is not restricted to the green-emissive TO but can be expanded to cyan and red. A new base surrogate (4,4-linked bisquinoline) provided up to 195-fold enhancement of the fluorescence.

Monitoring of RNA Dynamics in Living Cells Using PUM-HD and Fluorescent Protein Reconstitution Technique.

Fluorescence live-cell RNA imaging to monitor the intracellular localization and dynamics of the target RNA is a challenging subject. One of the difficulties to achieve this is to establish a precise method to enable a fluorescent labeling to the target RNA in living cells. Technologies to reduce the background fluorescence and to detect the RNA with high sensitivity are also necessary to visualize and analyze the intracellular localization and dynamic of the target RNA precisely. Especially in monitoring single-molecule motion, a special setup of a microscope system is required. Such technical problems make the live-cell RNA imaging to be a difficult subject. We recently developed a methodology to label and to visualize a target RNA in living cells with low background fluorescence by using a probe that is based on an RNA-binding protein domain PUM-HD (pumilio homology domain) and a fluorescent protein reconstitution method. A noteworthy property of PUM-HD to apply RNA probes is that this protein domain can be modified to recognize a particular 8-base RNA sequence by inducing tailor-made designed mutagenesis. The fluorescent protein reconstitution method allows us to detect the target RNA with high signal-to-noise ratio. Using the probe based on PUM-HD, a fluorescent protein reconstitution method, and a homebuilt fluorescent microscope system, we succeeded in single-molecule observation of a target RNA in living cells. In this chapter, the techniques to establish the probe and to observe the motion of single-molecule RNA are described.

Tandem Spinach Array for mRNA Imaging in Living Bacterial Cells.

Live cell RNA imaging using genetically encoded fluorescent labels is an important tool for monitoring RNA activities. A recently reported RNA aptamer-fluorogen system, the Spinach, in which an RNA aptamer binds and induces the fluorescence of a GFP-like 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) ligand, can be readily tagged to the RNA of interest. Although the aptamer–fluorogen system is sufficient for imaging highly abundant non-coding RNAs (tRNAs, rRNAs, etc.), it performs poorly for mRNA imaging due to low brightness. In addition, whether the aptamer-fluorogen system may perturb the native RNA characteristics has not been systematically characterized at the levels of RNA transcription, translation and degradation. To increase the brightness of these aptamer-fluorogen systems, we constructed and tested tandem arrays containing multiple Spinach aptamers (8–64 aptamer repeats). Such arrays enhanced the brightness of the tagged mRNA molecules by up to ~17 fold in living cells. Strong laser excitation with pulsed illumination further increased the imaging sensitivity of Spinach array-tagged RNAs. Moreover, transcriptional fusion to the Spinach array did not affect mRNA transcription, translation or degradation, indicating that aptamer arrays might be a generalizable labeling method for high-performance and low-perturbation live cell RNA imaging.

Hybridization-sensitive fluorescent oligonucleotide probe conjugated with a bulky module for compartment-specific mRNA monitoring in a living cell.

Live-cell RNA imaging at specific intracellular locations is technically limited because of the diffusive nature of small oligonucleotide probes. The bulky fluorescent light-up probes that possess streptavidin or gold nanoparticles at the end of oligonucleotides were designed and synthesized. The bulky probes allowed nucleus- and cytoplasm-selective monitoring of endogenous mRNAs through nuclear and cytoplasmic microinjection, respectively. Simultaneous use of bulky and unbulky probes conjugated with different fluorescent dyes enabled dual color imaging of mRNAs present in nucleus and cytoplasm. Furthermore, we observed that the fluorescence near the cell edge in a living HeLa cell traveled over time in coordination with the dynamic formation and deformation of the pseudopodial protrusions after lipofection of the bulky probes.

Live-cell imaging of mammalian RNAs with Spinach2.

The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag.

RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking.

Because RNA lacks strong intrinsic fluorescence, it has proven challenging to track RNA molecules in real time. To address this problem and to allow the purification of fluorescently tagged RNA complexes, we have selected a high affinity RNA aptamer called RNA Mango. This aptamer binds a series of thiazole orange (fluorophore) derivatives with nanomolar affinity, while increasing fluorophore fluorescence by up to 1,100-fold. Visualization of RNA Mango by single-molecule fluorescence microscopy, together with injection and imaging of RNA Mango/fluorophore complex in C. elegans gonads demonstrates the potential for live-cell RNA imaging with this system. By inserting RNA Mango into a stem loop of the bacterial 6S RNA and biotinylating the fluorophore, we demonstrate that the aptamer can be used to simultaneously fluorescently label and purify biologically important RNAs. The high affinity and fluorescent properties of RNA Mango are therefore expected to simplify the study of RNA complexes.

Understanding the Photophysics of the Spinach–DFHBI RNA Aptamer–Fluorogen Complex To Improve Live-Cell RNA Imaging

The use of aptamer-fluorogen complexes is an emerging strategy for RNA imaging. Despite its promise for cellular imaging and sensing, the low fluorescence intensity of the Spinach-DFHBI RNA aptamer-fluorogen complex hampers its utility in quantitative live-cell and high-resolution imaging applications. Here we report that illumination of the Spinach-fluorogen complex induces photoconversion and subsequently fluorogen dissociation, leading to fast fluorescence decay and fluorogen-concentration-dependent recovery. The fluorescence lifetime of Spinach-DFHBI is 4.0 ± 0.1 ns irrespective of the extent of photoconversion. We detail a low-repetition-rate illumination scheme that enables us to maximize the potential of the Spinach-DFHBI RNA imaging tag in living cells.

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5' end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.

A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging

A new fluorescent nucleotide with desmethyl thiazole orange dyes, D'(505), has been developed for expansion of the function of fluorescent probes for live-cell RNA imaging. The nucleoside unit of D'(505) for DNA autosynthesis was soluble in organic solvents, which made the preparation of nucleoside units and the reactions in the cycles of DNA synthesis more efficient. The dyes of D'(505)-containing oligodeoxynucleotide were protonated below pH 7 and the oligodeoxynucleotide exhibited hybridization-sensitive fluorescence emission through the control of excitonic interactions of the dyes of D'(505). The simplified procedure and effective hybridization-sensitive fluorescence emission produced multicolored hybridization-sensitive fluorescent probes, which were useful for live-cell RNA imaging. The acceptor-bleaching method gave us information on RNA in a specific cell among many living cells.

PNA FIT-Probes for the Dual Color Imaging of Two Viral mRNA Targets in Influenza H1N1 Infected Live Cells

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.

Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA), which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

Fluorescence Imaging of Influenza Virus H1N1 mRNA in Living Infected Cells using Single Chromophore FIT-PNA

Replication of the influenza virus involves, amongst other critical steps, the synthesis of viral mRNA which is used for ribosomal synthesis of viral proteins. Significant efforts have been devoted to the development of methods that allow the imaging of specific mRNA sequences in living cells. In contrast to other approaches, fluorescent oligonucleotide probes enable the analysis of non-modified wild-type targets. For imaging the probes must recognize the target with high specificity and deliver measurable signals that provide high signal-to-background ratios. We have introduced single labelled peptide nucleic acid (PNA) probes, so-called FIT-PNA probes, which contain a single thiazole orange intercalator that serves as artificial fluorescent nucleobase. These probes respond to changes of the local structure in the vicinity of the dye rather than to the more global changes of conformation that are required for fluorescence signalling by the dual labelled molecular beacons. FIT-probes are unique because a single fluorophore provides for both high sensitivity and high target specificity at non-stringent hybridization conditions where both matched and mismatched probe-target complexes coexist. We show that FIT-probes are ideally suited for applications in live cell RNA imaging. We chose mRNA coding for neuraminidase of influenza virus A/PR/8 as a target. FIT-PNA probes are useful to detect the expression of viral mRNA in single living infected cells with high specificity. In particular, FIT-PNA sensitivity was superior to that of a molecular beacon. Our study suggests that FIT-PNAs are attractive tools to study gene regulation of processes in living cells. The approach can be readily applied to other viruses as well.

Fluorescent Probes for Live-Cell RNA Detection

Commonly used techniques for analyzing gene expression, such as polymerase chain reaction (PCR), microarrays, and in situ hybridization, have proven invaluable in understanding RNA processing and regulation. However, these techniques rely on the use of lysed and/or fixed cells and are therefore limited in their ability to provide important spatial-temporal information. This has led to the development of numerous techniques for imaging RNA in living cells, some of which have already provided important insight into the dynamic role RNA plays in dictating cell behavior. Here we review the fluorescent probes that have allowed for RNA imaging in living cells and discuss their utility and limitations. Common challenges faced by fluorescent probes, such as probe design, delivery, and target accessibility, are also discussed. It is expected that continued advancements in live cell imaging of RNA will open new and exciting opportunities in a wide range of biological and medical applications.

Doubly Thiazole Orange-Labeled DNA for Live Cell RNA Imaging

Abstract A hybridization-sensitive, quencher-free fluorescent probe for RNA detection has been designed using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleoside showing high fluorescence intensity for a hybrid with the target RNA and effective quenching for the single-stranded state. The probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence from the probe hybridizing with the target RNA was observed. This fluorescence rapidly decreased upon addition of a competitor DNA. This probe realized a large, rapid, and reversible change in fluorescence intensity in sensitive response to the amount of target RNA, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.