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Developing a Fluorescent Toolbox To Shed Light on the Mysteries of RNA Seth C. Alexander and Neal K. Devaraj* Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States ABSTRACT: Technologies that detect and image RNA have illuminated the complex roles played by RNA, redefining the traditional and superficial role first outlined by the central dogma of biology. Because there is such a wide diversity of RNA structure arising from an assortment of functions within biology, a toolbox of approaches have emerged for investigation of this important class of biomolecules. These methods are necessary to detect and elucidate the localization and dynamics of specific RNAs and in doing so unlock our understanding of how RNA dysregulation leads to disease. Current methods for detecting and imaging RNA include in situ hybridization techniques, fluorescent aptamers, RNA binding proteins fused to fluorescent reporters, and covalent labeling strategies. Because of the inherent diversity of these methods, each approach comes with a set of strengths and limitations that leave room for future improvement. This perspective seeks to highlight the most recent advances and remaining challenges for the wide-ranging toolbox of technologies that illuminate RNA’s contribution to cellular complexity. readout,9 the development of more efficient DNA synthesis technologies has permitted the creation of longer (30−90nucleotide) antisense oligonucleotides bearing fluorophores directly conjugated to the ends of these probes. Once fluorescent modified antisense probes could be readily synthesized, RNA fluorescence in situ hybridization (RNAFISH) made imaging of RNA a much more accessible technique compatible with standard fluorescence microscopy.8 In RNA-FISH, designed antisense sequences selectively anneal target endogenous RNAs within fixed tissues and, in doing so, reveal the RNA’s locale within the cell (Figure 2A). While this approach is sufficient for imaging a wide variety of RNAs, it typically requires tiling of multiple fluorophores within a single RNA target to obtain single-molecule resolution10 and therefore can be impractical for shorter and less abundant RNA targets. Additionally, off-target binding and the inability to discriminate between single-nucleotide mismatches limit imaging studies by this approach. Improvements in the signal-to-noise ratio can be achieved with the use of molecular beacons, which harness the power of proximity quenching through a hairpin structure that masks fluorescence until the molecular beacon is hybridized to its proper target. Once bound to its target, the quencher and fluorophore are separated, leading to an increase in fluorescence.11 In a similar fashion, two adjacent Förster resonance energy transfer (FRET) donor and acceptor hybridizing probes can be used to provide an improvement in both signal-to-noise ratio and specificity.12
RNA is the cornerstone of biology’s central dogma, orchestrating a highly complex collection of functions critical to the regulation of numerous cellular pathways and processes.1 However, when compared to our understanding of proteins and chromosomes, our understanding of RNA’s diverse cellular roles is significantly lacking, in part because of the transient nature of RNA and the challenges associated with visualizing low-copy number (as low as one copy per cell2) RNAs and accurately mapping their life history from synthesis to degradation. While RNA was initially thought to serve only as a message from the instructional code of DNA to perform the synthesis of proteins, it has become increasingly apparent that RNA contributes equally with its protein counterparts to cellular complexity, especially with respect to disease.3 While methods so far developed have made a significant impact in shedding light on the mysteries of RNA, tools are still needed to pierce deeper behind the veil of the cell and illuminate the diverse functional contributions of RNA to life. In this perspective, we aim to address the most recent and exciting technological developments in detecting and visualizing RNA by a variety of approaches, including in situ hybridization techniques,4 fluorescent aptamers,5 RNA binding proteins fused to fluorescent reporters,6 and covalent labeling strategies7 (Figure 1). While this work does not aim to be comprehensive, we seek to highlight significant advancements in the past few years for RNA imaging and provide insight for the future of the field.
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IN SITU HYBRIDIZATION TECHNIQUES Nucleic acid antisense in situ hybridization techniques are a mainstream and well-established method for visualizing and detecting RNA in fixed tissues and in vitro samples.4,8 While this method was first reported using an immunofluorescent © XXXX American Chemical Society
Special Issue: Seeing Into Cells Received: May 26, 2017 Revised: July 1, 2017 Published: July 3, 2017 A
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Figure 1. Overview of current approaches for fluorescent detection and imaging of RNA.
probes that greatly improved binding and specificity through constraints of the nucleic acid backbone, dramatically increasing the impact FIT probes have made on RNA detection and imaging.20,21 In a similar fashion, Okamoto incorporated TO derivatives into locked nucleic acid (LNA) and DNA excitoncontrolled hybridization-sensitive fluorescent oligonucleotide (ECHO) probes to detect DNA and RNA sequences in a similar fashion.22,23 FIT and ECHO probes not only serve as fluorogenic antisense probes of RNA but also have been employed to image RNA using wash free conditions24 and have been most recently expanded to include multicolor RNA imaging via a red light-emitting (605 nm) quinolone blue FIT probe.25 Harnessing in situ hybridization to catalyze chemical transformations has further diversified methods that discriminate single-nucleotide mismatches and provides a quantitative readout of specific DNA and RNA levels within samples. For example, seminal work by the Kool group demonstrated the use of a nucleic acid-templated reaction that releases a quencher from its neighboring fluorophore to restore fluorescence only upon perfect complementation with a target sequence.26 Since that time, many groups have explored a variety of ligation and transfer reactions templated by DNA and RNA to achieve specific detection.27−29 Recently, our lab has employed a variety of tetrazine ligation and transfer reactions that result in large increases in the intensity of the fluorescent signal upon hybridization with DNA and RNA target sequences (Figure 2C).30 Initially, we explored a ligation reaction between a small cyclopropene dienophile and a BODIPY-FL quenched tetrazine moiety that gave an ∼10-fold increase in fluorescence upon oligonucleotide-templated ligation. Detection by this method was limited because of the ligation reaction, which creates a tighter binding antisense probe and prevents multiple turnovers of the reactive probes for signal amplification. By employing an
Development of branching scaffolds has allowed for detection and imaging of single RNAs by providing numerous binding sites for fluorescent probes per target binding site (Figure 2B), thus amplifying fluorescent signal output per single binding event without the need for tiling many different synthetically expensive probes.13 Using this technique, single-molecule RNA imaging to quickly define transcriptome-wide RNA localization in a high-throughput fashion has been recently achieved.14 Despite these advances, however, off-target labeling and limitations of delivery of a probe to live tissues remain the most significant concerns that practitioners must keep in mind when employing these approaches. While it is important to visualize the locale of specific RNAs within tissues, the detection of single-nucleotide differences within nucleic acid sequences is extremely important. Specifically, the ability to detect single-nucleotide polymorphisms (SNPs), especially for miRNAs, has a significant diagnostic value for early detection of diseases such as cancer.15,16 In situ hybridization techniques have been developed to detect and quantify specific RNAs by a fluorescent readout.17 While FRET or molecular beacon antisense approaches can provide a detectable signal upon hybridization to specific nucleic acid sequences, the Seitz group pioneered an early sensitive approach that harnessed peptide nucleic acid (PNA) conjugates bearing thaizole orange (TO) fluorophores that would be forced to intercalate (FIT) upon hybridization with a target nucleic acid sequence. PNA-FIT probes thus provide a fluorogenic response when hybridized to a target nucleic acid sequence through specific interaction between the TO and stacked bases. Because of sensitive environmental changes, FIT probes can detect single neighboring mismatches near the site of intercalation, a critical factor in detecting SNPs effectively.18,19 Expanding the FIT probe architecture, the Seitz group has also employed locked nucleic acid (LNA) FIT B
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Figure 2. In situ hybridization approaches. (A) RNA-FISH and variants using molecular beacons and FRET probes. (B) DNA branched FISH. (C) Click-to-release turn-on hybridization probes. (D) Nano-flares. Panel C adapted with permission from ref 33. Copyright 2016 American Chemical Society. Panel D reprinted with permission from ref 38. Copyright 2014 National Academy of Sciences.
fluorescent reporter sequences that are efficiently quenched by hybridization to antisense strands conjugated to a gold nanoparticle. Once they are delivered into live cells, binding of the antisense strand with the target mRNA results in displacement and release of the fluorophore-labeled strands and an increase in fluorescence that is quantitatively proportional to the concentration of endogenous mRNA target (Figure 2D). Over the past few years, this technology has rapidly developed, improving many of its original drawbacks. Incorporation of an internal FRET pair within the reporter oligonucleotide (FRET nano-flares) has significantly improved false positive signals imparted by thermodynamic fluctuations and biochemical interference such as degradation from nucleases.36 Additionally, the development of sticky-flares has transformed this technology from an RNA detection method to a quantitative imaging technique by making the reporter “flare” complementary to the target RNA such that the fluorescent reporter anneals to the target and reports on its location.37 Recent applications include detection and sorting of circulating cancer cells in human blood samples38 and adaptation of a similar FRET nanoparticle approach by the Tang lab that allowed for simultaneous multiplex detection of mRNA cancer markers in live cells.39 These recent applications demonstrate the robustness and applicability of this technology in oncology and related diagnostic point-of-care field applications.
alternative dienophile, 7-azabenzonorbornadiene, ligation was subsequently followed by a spontaneous cleavage reaction, permitting strand displacement and multiple reactions per template strand. This turnover amplification resulted in a >100fold fluorescent turn-on and improved the limit of detection to as low as 5 pM target RNA.31 Strong quenching of the BODIPY-FL moiety by tetrazines is achieved via FRET between the broad spectral overlap of green fluorphores’ emission and the absorbance of an adjacent tetrazine moiety.32 Therefore, this method works optimally with green fluorescent reporters. We recently overcame this spectral limitation by employing an alternative click-to-release mechanism between vinyl ether-caged fluorophores, including a near-infrared (nearIR) Cy5.5 fluorescent reporter and tetrazines by way of internal charge transfer (ICT) quenching.33 By employing a vinyl ether cage on a key alcohol group of Cy5.5 or a coumarin derivative, fluorescence remains quenched until the vinyl group is cleaved via cycloaddition with a tetrazine followed by subsequent decomposition. Harnessing this second method of fluorogenic reactivity has significantly expanded the palette of fluorescent reporters that can be used by this approach to detect DNA and RNA both in vitro and in live cells.33 Most hybridization-based detection methods require either fixed tissues, the use of transfection agents, or microinjection. However, the development of a gold nanoparticle spherical nucleic acid (SNA) as a delivery vehicle has greatly expanded the use of live tissue detection of RNAs. SNAs provide a unique architecture for nucleic acid delivery and do not require the use of cytotoxic transfection agents for live cell applications but rather are readily taken up by cells through an endocytotic pathway.34 First conceptualized by the Mirkin group in 2007,35 SNAs capable of reporting on RNA levels via fluorescence, also known as nano-flares, take advantage of hybridizing short
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RNA APTAMER SYSTEMS Fluorogenic RNA aptamers have recently become a powerful method for imaging specific RNAs in live cells. These systems work by encoding specific sequences within a target RNA to impart a modular RNA binding domain for pro-fluorescent small molecules. The encoded aptamer domain, when bound to C
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Figure 3. RNA binding proteins fused to fluorescent reporters. (A) MS2 and related hairpin binding proteins fused to fluorescent proteins or split fluorescent proteins for the visualization of a specific RNA. (B) rCas9-GFP targeting of natural RNA without the need to encode recognition elements. Panel A adapted with permission from ref 6. Copyright 2015 Macmillan Publishers Ltd. Panel B adapted with minimal changes with permission from ref 69. Copyright 2016 Elsevier.
its respective small molecule, elicits an increase in fluorescence that can be visualized to report on the location of the aptamer encoding RNA. While RNA aptamers have been known to bind small molecules since 1990,40,41 it was not until 2003 that the Tsien lab reported the first fluorogenic aptamers of malachite green (MG) and sulforhodamine B (SRB) for applications in RNA detection and imaging. Tsien and co-workers reported the MG aptamer bound its substrate with a binding affinity of 117 nM that resulted in an impressive >2000-fold increase in quantum yield in vitro. Unfortunately, because of the toxicity of MG within live cells, this aptamer was limited to in vitro applications. In 2011, Jaffrey and co-workers reported a live cell compatible 98-nucleotide RNA mimic of GFP, called Spinach, that bound an analogue of GFP’s chromophore, 3,5-difluoro-4hydroxybenzylidene imidazolinone (DFHBI). Spinach was found to bind DFHBI with an affinity of 537 nM and a brightness that is roughly half that of EGFP. They successfully demonstrated that the aptamer could report on the location of expressed RNA−Spinach fusions in live cells.42 Spinach was initially limited to a single-color chromophore and suffered from low brightness in cells when appended to target RNAs due to thermal instability and poor folding. These limitations were overcome by an optimized “superfolding” Spinach II aptamer,43 as well as demonstration of a plug-and-play set of derivatives of the HBI fluorogenic core that widened the color palette for this aptamer-based imaging approach.44 Recent developments such as Broccoli45 and other shorter (1000-fold increase in fluorescence intensity upon complex formation. The exceptionally high affinity for TO1-biotin permits the effective streptavidin-based purification of RNAs containing the short 39-nucleotide Mango motif. Although imaging is likely possible with this approach, it is unclear how well it will fare with low-abundance targets because of the high fluorescent background of TO within cellular environments. While recently developed aptamers have opened up the possibility of live cell imaging of RNA, they do not come without a key set of limitations to be considered, most notably, the requirement to encode a >50-nucleotide RNA structure that must properly fold, limiting this approach to applications for RNAs that do not have rigid sequence length or structural requirements altered by encoding the reporter aptamer. Additionally, a pro-fluorescent small molecule must also be delivered to the cellular locale of the aptamer to achieve binding and fluorescence visualization. Future directions of this field will undoubtedly focus on the development of smaller, brighter aptamers, as well as new aptamers that provide near-IR and super-resolution imaging capabilities.
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RNA BINDING PROTEINS Functional RNA within cells is typically bound to one or more RNA binding proteins (RBPs). It is therefore perhaps unsurprising that RBPs, when fused to fluorescent reporters such as GFP, are effective tools for imaging RNA in live cells. The RNA phage capsid protein MS2 (also known as R17) was first harnessed to act as a purification method for RNAs bearing a short hairpin binding motif.56 It was soon thereafter D
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COVALENT LABELING STRATEGIES While many excellent noncovalent methods exist to label, detect, and image RNA in fixed and living systems, covalent approaches have remained challenging because of difficulties in selectively modifying one transcript among the diverse pool of RNA found within the cell. Covalent strategies provide an additional level of robustness and irreversibility that simply cannot be obtained by noncovalent approaches. There are many situations, such as targeting low-abundance RNA, in which reversible dissociation of the reporter from its target can be of significant concern and lead to potential artifacts. Additionally, binding large proteins or long hybridization probes to target RNAs can alter their structure, function, processing, and, therefore indirectly, localization within the cell. Via conjugation of a relatively low-molecular weight fluorophore (150 kDa) of Cas9, it is likely that the protein’s bulk will influence the processing and regulation of bound RNAs. As discussed previously, this is already a concern for RNAs bound to the much smaller MS2 fusions. Ideally, future improvements that reduce the size of these reporters while allowing the targeting of native RNA will overcome current limitations. E
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Figure 4. RNA covalent labeling strategies. (A) Methyltransferase Tgs harnessing click handle AdoMet analogues as substrates to label the 5′ cap of mRNAs. Secondary reactions with fluorescent click partners can be used to report on the location of mRNA. (B) Dual 5′ cap labeling of mRNA based on regioselective methyltransferases and bioorthogonal chemistry to improve the signal-to-noise ratio for imaging applications. (C) Covalent modification of archaeal tRNA fused to an RNA of interest by the tRNA-modifying enzyme Tias. Click handles can be incorporated and then labeled with fluorophores in a second step to report on the location of RNA in fixed tissue. (D) RNA-TAG platform to covalently label RNA directly with fluorophores and affinity agents. Encoding of a small hairpin into an RNA of interest allows for the selective direct incorporation of preQ1 conjugated fluorophores and affinity agents by the Escherichia coli tRNA-modifying enzyme, TGT. Panel A reproduced in part with permission from ref 72. Copyright 2013 John Wiley & Sons, Inc. Panel D reprinted with permission from ref 77. Copyright 2015 American Chemical Society.
site-specific labeling to occur. By genetically encoding the archaeal tRNA to an RNA of interest, target RNAs could be site-specifically labeled by this approach and, via a secondary click reaction, provide a method for RNA imaging in fixed tissues (Figure 4C). However, the requirement of millimolar concentrations of propargyl amine or a short amino-alkyl azide undoubtedly will preclude its adaptation to live cell imaging, and the presence of competing short primary amines found in cells could prevent low-abundance RNAs from being labeled to a detectable level. Additionally, the need to encode an entire tRNA (77 nucleotides) imposes a limitation on the size and types of RNA that would not be affected by addition of such a relatively long and biologically common sequence and structure. Taking a cue from the diversity of tRNA-modifying enzymes, our group has recently harnessed a bacterial tRNA guanine transglycosylase (TGT) to conjugate functional reporters such as fluorophores and affinity handles site-specifically to an RNA of interest in a single enzymatic step.77 Escherichia coli TGT has been extensively characterized and is known to natively incorporate the nucleobase preQ1 into the G34 wobble
after bioorthogonal conjugation or allows for the separate introduction of both a fluorophore and an affinity handle on each bioconjugated mRNA (Figure 4B).75 However, while this method is excellent for labeling mRNA transcripts, its approach is limited to labeling at the 5′ end of an RNA and still lacks selectivity for a specific transcript among members of the specific class of RNA that bear the traditional 5′ cap. tRNAs are well-known to undergo enzymatic post-transcriptional modifications, and as such, there has been significant interest in adapting tRNA modification enzymes for covalent modification of RNA. tRNA-modifying enzymes have shown promise in complementing current technologies for imaging RNA by covalent means. Wang and co-workers demonstrated that the tRNA-modifying enzyme Tias could site-specifically and covalently append its primary amine substrate to the C34 wobble position within the anticodon stem loop of archaeal tRNAIle2.76 Archaeal Tias naturally incorporates the short primary amine agmatine, and its RNA recognition requirements are orthogonal to eukaryotic systems. Because of the inherent promiscuity of this enzyme, small amines bearing alkynes and azides were tolerable substrates for the enzyme and allowed F
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covalent attachment is currently one of the most attractive ways to overcome these limitations, much development is still needed to make covalent strategies attractive over more wellestablished approaches. Because of the diversity in class, size, and function of RNAs within cells, continuing to expand the toolbox of approaches will be critical to unlocking the deeper mysteries of RNA.
position of tRNAs that bear a UGU in their anticodon stem loop.78,79 E. coli TGT is unique in that it does not require the entire tRNA structure for substrate recognition but instead can efficiently recognize the anticodon stem loop region of the tRNA, even when encoded into other RNA sequences. E. coli TGT is exclusive and distinctive from its eukaryotic counterpart, both in small molecule substrate recognition (queuine vs preQ1), as well as in RNA recognition requirements (whole tRNA vs hairpin alone). These key differences provide a unique condition that allows labeling by E. coli TGT to remain selective for a short encoded hairpin. While extensive selectivity studies have yet to be performed, we would expect few offtarget sites to be labeled to any significant extent because of both sequence and structural requirements as outlined in past work.79 PreQ1-conjugated fluorophores of BODIPY-FL, Cyanine-7, and a fluorogenic TO have all been directly incorporated by E. coli TGT into an expressed RNA transcript bearing the recognition stem loop (Figure 4D).77 We found that RNA transglycosylation at guanosine, or RNA-TAG, could be used to image a model cellular mRNA in fixed cells. We also demonstrated the ability to functionally label and isolate a target transcript by conjugating a biotin preQ1 derivative and selectively purifying the labeled transcript from total RNA extract with streptavidin-coated magnetic beads. While this approach may lead to powerful imaging applications, another asset is the ability to directly conjugate a wide variety of functional handles beyond fluorescent reporters to study, characterize, and further derivatize RNA. Although our initial proof-of-concept application did not include live cell labeling, we are currently adapting RNA-TAG to be compatible with live cell imaging and labeling workflows. Although site-specific covalent labeling has only recently been developed as a strategy for RNA imaging and detection, these approaches show great promise for labeling individual RNAs with a variety of reporters. Although single modifications limit the detection of low-abundance targets, development of additional techniques that amplify these modifications can easily overcome this current limitation. The robust and diverse advantage of covalent modifications is undeniable, and it is clear that researchers will continue to develop and improve covalent labeling technologies to complement existing noncovalent approaches.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Neal K. Devaraj: 0000-0002-8033-9973 Funding
This material is based upon work supported by the Army Research Office through the MURI program under Award No. W911NF-13-1-0383. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the thoughtful insights of Professor Brian Zid as well as helpful editing from Kayla N. Busby. ABBREVIATIONS FISH, fluorescence in situ hybridization; SNP, single-nucleotide polymorphism; PNA, peptide nucleic acid; TO, thiazole orange; FIT, forced to intercalate; ECHO, excitation-controlled hybridization-sensitive fluorescent oligonucleotide; LNA, locked nucleic acid; ICT, internal charge transfer; SNA, spherical nucleic acid; FRET, Fö rster resonance energy transfer; MG, malachite green; SRB, sulforhodamine B; DFHBI, 3,5-difluoro-4-hydroxybenzylidene imidazolinone; RBP, RNA binding protein; AdoMet, 5-adenosyl-1-methionine; TGT, tRNA guanine transglycosylase.
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REFERENCES
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CONCLUDING REMARKS The importance of RNA and its localization continues to become more apparent the deeper we dive into the complexity of the cell. It is clear an ever-expanding and -diversifying toolbox of approaches to visualize and detect these dynamic molecules in cells is critical to obtaining a complete and accurate picture of the many roles of RNA in biology. Current methods each come with not only a set of strengths but also limitations and caveats that can be only partially overcome by investigations using complementary approaches. While many of the current approaches have been shown to elucidate several aspects of RNA biology, established techniques are still largely limited to high-abundance RNA targets. Continuing refinement of methods that allow imaging of low concentrations of RNA, down to single-copy RNAs within a cell, is still needed. Additionally, because of the dynamic nature of RNA, methods that not only can visualize RNA in live cells but also can report on the dynamic nature of RNA in a way that is minimally perturbing to its natural life remain a significant challenge. While introduction of small fluorescent reporters through G
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