Fluorogenic RNA Aptamers Research Articles

Single mRNA Imaging with Fluorogenic RNA Aptamers and Small-molecule Fluorophores.

Messenger RNA (mRNA) is the fundamental information transfer system in the cell. Tracking single mRNA from transcription to degradation with fluorescent probes provides spatiotemporal information in cells about how the genetic information is transferred from DNA to proteins. The traditional single mRNA imaging approach utilizes RNA hairpins (e.g. MS2) and tethered fluorescent protein as probes. As an exciting alternative, RNA aptamers: small-molecule fluorophores (SFs) systems have emerged as novel single mRNA imaging probes since 2019, exhibiting several advantages including fluorogenic ability and minimal perturbation. This review summarizes all five reported RNA aptamers: SFs systems for single mRNA imaging in living cells so far. It also discusses the challenges and provides prospects for single mRNA imaging applications. This review is expected to inspire researchers to develop RNA aptamers: SFs systems for studying gene expression at single-molecule resolution in cells.

Probing of Fluorogenic RNA Aptamers via Supramolecular Förster Resonance Energy Transfer with a Universal Fluorescent Nucleobase Analog.

Fluorogenic RNA aptamers are synthetic RNAs that have been evolved by in vitro selection methods to bind and light up conditionally fluorescent organic ligands. Compared with other probes for RNA detection, they are less invasive than hybridization-based methods (FISH, molecular beacons) and are considerably smaller than fluorescent protein-recruiting systems (MS2, Pumilio variants). Fluorogenic aptamers have therefore found widespread use as genetically encodable tags for RNA detection in live cells and have also been used in combination with riboswitches to construct versatile metabolite sensors for in vitro use. Their success builds on a fundamental understanding of their three-dimensional structure to explain the mechanisms of ligand interaction and to rationally design functional aptamer devices. In this protocol, we describe a supramolecular FRET-based structure probing method for fluorogenic aptamers that exploits distance- and orientation-dependent energy transfer efficiencies between site-specifically incorporated fluorescent nucleoside analogs and non-covalently bound ligands, exemplified by 4-cyanoindol riboside (4CI) and the DMHBI+-binding RNA aptamer Chili. This method yields structural restraints that bridge the gap between traditional low-resolution secondary structure probing methods and more elaborate high-resolution methods such as X-ray crystallography and NMR spectroscopy.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers.

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s-1 and 0.35 s-1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

Structural Basis for Fluorescence Activation by Pepper RNA.

Pepper is a fluorogenic RNA aptamer tag that binds to a variety of benzylidene-cyanophenyl (HBC) derivatives with tight affinity and activates their fluorescence. To investigate how Pepper RNA folds to create a binding site for HBC, we used antibody-assisted crystallography to determine the structures of Pepper bound to HBC530 and HBC599 to 2.3 and 2.7 Å resolutions, respectively. The structural data show that Pepper folds into an elongated structure and organizes nucleotides within an internal bulge to create the ligand binding site, assisted by an out-of-plane platform created by tertiary interactions with an adjacent bulge. As predicted from a lack of K+ dependence, Pepper does not use a G-quadruplex to form a binding pocket for HBC. Instead, Pepper uses a unique base-quadruple·base-triple stack to sandwich the ligand with a U·G wobble pair. Site-bound Mg2+ ions support ligand binding structurally and energetically. This research provides insight into the structural features that allow the Pepper aptamer to bind HBC and show how Pepper's function may expand to allow the in vivo detection of other small molecules and metals.

Self-Assembly of Intracellular Multivalent RNA Complexes Using Dimeric Corn and Beetroot Aptamers.

DNA and RNA can spontaneously self-assemble into various structures, including aggregates, complexes, and ordered structures. The self-assembly reactions cannot be genetically encoded to occur in living mammalian cells since the double-stranded nucleic acids generated by current self-assembly approaches are unstable and activate innate RNA immunity pathways. Here, we show that recently described dimeric aptamers can be used to create RNAs that self-assemble and create RNA and RNA-protein assemblies in cells. We find that incorporation of five copies of Corn, a dimeric fluorogenic RNA aptamer, into an RNA causes the RNA to form large clusters in cells, reflecting multivalent RNA-RNA interactions enabled by these RNAs. Here, we also describe a second dimeric fluorogenic aptamer, Beetroot, which shows partial sequence similarity to Corn. Both Corn and Beetroot form homodimers with themselves but do not form Corn-Beetroot heterodimers. We thus use Corn and Beetroot to encode distinct RNA-protein assemblies in the same cells. Overall, these studies provide an approach for inducing RNA self-assembly, enable multiplexing of distinct RNA assemblies in cells, and demonstrate that proteins can be recruited to RNA assemblies to genetically encode intracellular RNA-protein assemblies.

Multiplexed miRNA detection based on target-triggered transcription of multicolor fluorogenic RNA aptamers

We herein describe a new multicolor fluorogenic RNA aptasensor to accomplish multiplexed detection of miRNAs. The stem-loop primer (SL primer) entailing a fluorogenic RNA aptamer (FRA) antisense sequence is designed to anneal to target miRNA at its 3′ overhang, which would be reverse transcribed by reverse transcriptase (RT) to produce the cDNA sequence followed by the degradation of target miRNA. The T7 promoter-containing primer (T7 primer) is then annealed to the 3’ end of the extended cDNA sequence and the following RT-promoted extension in both directions produces the T7 promoter-containing double-stranded DNA (T7 dsDNA). T7 RNA polymerase finally transcribes the T7 dsDNA to produce a large number of RNA transcripts containing FRA sequence, which would produce intense fluorescence signals by forming fluorescent complexes with cognate fluorogens, reflecting the amount of target miRNAs. Based on this unique design principle employing the SL primers to encode several different FRAs with distinct fluorescence profiles, target miRNAs were very specifically determined in a multiplexed manner down to a subpicomolar level. The practical applicability of this technique was also verified by reliably quantifying target miRNAs in serum and human cancer cell lysates.

RNA Mango: a versatile tool to track cellular RNAs and for rapid viral RNA detection

In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper, have gathered traction as an efficient alternative labelling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which have improved both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs in mammalian cells with single-molecule sensitivity.

Fluorogenic RNA aptamers to probe transcription initiation and co-transcriptional RNA folding by multi-subunit RNA polymerases.

Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of biochemical processes. Green fluorescent proteins have long served as valuable tools for studying the process of translation. Here we present two methods that utilize fluorescent light-up RNA aptamers (FLAPs), the RNA mimics of green fluorescent proteins, to monitoring transcription and co-transcriptional RNA folding. FLAPs adopt defined three-dimensional folds that bind low molecular weight compounds called fluorogens with concomitant increase in fluorescence by many folds. FLAPs provide a strong fluorescence signal with low background that allows monitoring of transcription in real time in vitro and in vivo. However, it takes several seconds for RNA polymerase to synthesize FLAPs and the subsequent folding of the fluorogen-binding platform takes additional seconds or minutes. Here we show that Broccoli-FLAP is well suited for monitoring the rate of transcription initiation in a multi-round setup that mitigates the slow rate of the FLAP maturation. Furthermore, we demonstrate that a relatively slow and inefficient folding of iSpinach-FLAP can be taken advantage of for monitoring the action of RNA folding chaperones.

Transcriptionally amplified synthesis of fluorogenic RNA aptamers for label-free DNA glycosylase assay.

We demonstrate for the first time the utilization of fluorogenic RNA aptamers for label-free uracil-DNA glycosylase (UDG) assay. Through rationally engineering the transcription machine with dU substitution, this assay requires only a single probe to simultaneously sense and amplify the UDG signal, achieving a low detection limit of 6.3 × 10-6 U mL-1. Moreover, it can be applied for screening UDG inhibitors and measuring endogenous UDG activity in different cells.

A fluorogenic RNA aptamer nanodevice for the low background imaging of mRNA in living cells.

A fluorogenic RNA aptamer nanodevice integrating an entropy-driven RNA amplifier with near-infrared (NIR) light control was developed, affording high contrast and sensitivity for imaging low-abundance mRNA in living cells. The design principle offers a new approach for developing low-background imaging systems for live-cell studies and manipulation.

Repurposing an adenine riboswitch into a fluorogenic imaging and sensing tag.

Fluorogenic RNA aptamers are used to genetically encode fluorescent RNA and to construct RNA-based metabolite sensors. Unlike naturally occurring aptamers that efficiently fold and undergo metabolite-induced conformational changes, fluorogenic aptamers can exhibit poor folding, which limits their cellular fluorescence. To overcome this, we evolved a naturally occurring well-folded adenine riboswitch into a fluorogenic aptamer. We generated a library of roughly 1015 adenine aptamer-like RNAs in which the adenine-binding pocket was randomized for both size and sequence, and selected Squash, which binds and activates the fluorescence of green fluorescent protein-like fluorophores. Squash exhibits markedly improved in-cell folding and highly efficient metabolite-dependent folding when fused to a S-adenosylmethionine (SAM)-binding aptamer. A Squash-based ratiometric sensor achieved quantitative SAM measurements, revealed cell-to-cell heterogeneity in SAM levels and revealed metabolic origins of SAM. These studies show that the efficient folding of naturally occurring aptamers can be exploited to engineer well-folded cell-compatible fluorogenic aptamers and devices.

Single-Molecule RNA Imaging Using Mango II Arrays.

In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.

Live‐Cell Imaging of Guanosine Tetra‐ and Pentaphosphate (p)ppGpp with RNA‐based Fluorescent Sensors**

AbstractGuanosine tetra‐ and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA‐based fluorescent sensors for the live‐cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp‐specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell‐to‐cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA‐based sensors can be broadly adapted to study bacterial stringent response.

Live-Cell Imaging of Guanosine Tetra- and Pentaphosphate (p)ppGpp with RNA-based Fluorescent Sensors*.

Guanosine tetra- and pentaphosphate, (p)ppGpp, are important alarmone nucleotides that regulate bacterial survival in stressful environment. A direct detection of (p)ppGpp in living cells is critical for our understanding of the mechanism of bacterial stringent response. However, it is still challenging to image cellular (p)ppGpp. Here, we report RNA-based fluorescent sensors for the live-cell imaging of (p)ppGpp. Our sensors are engineered by conjugating a recently identified (p)ppGpp-specific riboswitch with a fluorogenic RNA aptamer, Broccoli. These sensors can be genetically encoded and enable direct monitoring of cellular (p)ppGpp accumulation. Unprecedented information on cell-to-cell variation and cellular dynamics of (p)ppGpp levels is now obtained under different nutritional conditions. These RNA-based sensors can be broadly adapted to study bacterial stringent response.

Engineering Fluorophore Recycling in a Fluorogenic RNA Aptamer

AbstractFluorogenic aptamers can potentially show minimal photobleaching during continuous irradiation since any photobleached fluorophore can exchange with fluorescent dyes in the media. However, fluorophores have not been designed to maximize “fluorophore recycling.” Here we describe TBI, a novel fluorophore for the Broccoli fluorogenic aptamer. Previous fluorophores either fail to rapidly dissociate when they undergo photobleaching via cis–trans isomerization, or bind slowly, resulting in extended periods after dissociation of the photobleached fluorophore when no fluorophore is bound. By contrast, photobleached TBI dissociates rapidly from Broccoli, and TBI from the media rapidly replaces dissociated photobleached fluorophore. Using TBI, Broccoli exhibits markedly enhanced fluorescence in cells during continuous imaging. These data show that designing fluorophores to optimize fluorophore recycling can lead to enhanced fluorescence of fluorogenic aptamers.

Engineering Fluorophore Recycling in a Fluorogenic RNA Aptamer.

Fluorogenic aptamers can potentially show minimal photobleaching during continuous irradiation since any photobleached fluorophore can exchange with fluorescent dyes in the media. However, fluorophores have not been designed to maximize "fluorophore recycling." Here we describe TBI, a novel fluorophore for the Broccoli fluorogenic aptamer. Previous fluorophores either fail to rapidly dissociate when they undergo photobleaching via cis-trans isomerization, or bind slowly, resulting in extended periods after dissociation of the photobleached fluorophore when no fluorophore is bound. By contrast, photobleached TBI dissociates rapidly from Broccoli, and TBI from the media rapidly replaces dissociated photobleached fluorophore. Using TBI, Broccoli exhibits markedly enhanced fluorescence in cells during continuous imaging. These data show that designing fluorophores to optimize fluorophore recycling can lead to enhanced fluorescence of fluorogenic aptamers.

Large Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine

Fluorogenic RNA aptamers are synthetic functional RNAs that specifically bind and activate conditional fluorophores. The Chili RNA aptamer mimics large Stokes shift fluorescent proteins and exhibits high affinity for 3,5-dimethoxy-4-hydroxybenzylidene imidazolone (DMHBI) derivatives to elicit green or red fluorescence emission. Here, we elucidate the structural and mechanistic basis of fluorescence activation by crystallography and time-resolved optical spectroscopy. Two co-crystal structures of the Chili RNA with positively charged DMHBO+ and DMHBI+ ligands revealed a G-quadruplex and a trans-sugar-sugar edge G:G base pair that immobilize the ligand by π-π stacking. A Watson-Crick G:C base pair in the fluorophore binding site establishes a short hydrogen bond between the N7 of guanine and the phenolic OH of the ligand. Ultrafast excited state proton transfer (ESPT) from the neutral chromophore to the RNA was found with a time constant of 130 fs and revealed the mode of action of the large Stokes shift fluorogenic RNA aptamer.

Rapid Clinical Diagnostic Viral Detection with Saliva by a Novel Single Step Nested Mango-NASBA Assay

The recent COVID-19 pandemic has clearly highlighted the necessity for rapid and fieldable diagnostic coronavirus tests. However, few such methods meet all requirements of rapidity, sensitivity, and affordability, which hiders the prevention of uncontrolled widespread transmission of SARS-Cov-2. Here, we report a new rapid clinical diagnostic test for viral RNA detection using a recently developed fluorogenic RNA aptamer, Mango. This assay is based on an isothermal amplification of ribonucleic acid termed NASBA (nucleic acid sequence-based amplification).

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Imaging Intracellular S-Adenosyl Methionine Dynamics in Live Mammalian Cells with a Genetically Encoded Red Fluorescent RNA-Based Sensor.

To understand the role of intracellular metabolites in cellular processes, it is important to measure the dynamics and fluxes of small molecules in living cells. Although conventional metabolite sensors composed of fluorescent proteins have been made to detect some metabolites, an emerging approach is to use genetically encoded sensors composed of RNA. Because of the ability to rapidly generate metabolite-binding RNA aptamers, RNA-based sensors have the potential to be designed more readily than protein-based sensors. Numerous strategies have been developed to convert the green-fluorescent Spinach or Broccoli fluorogenic RNA aptamers into metabolite-regulated sensors. Nevertheless, red fluorescence is particularly desirable because of the low level of red background fluorescence in cells. However, the red fluorescent variant of the Broccoli aptamer, Red Broccoli, does not exhibit red fluorescence in cells when imaged with its cognate fluorophore. It is not known why Red Broccoli is fluorescent in vitro but not in live mammalian cells. Here, we develop a new fluorophore, OBI (3,5-difluoro-4-hydroxybenzylidene-imidazolinone-2-oxime-1-benzoimidazole), which binds Red Broccoli with high affinity and makes Red Broccoli resistant to thermal unfolding. We show that OBI enables Red Broccoli to be readily detected in live mammalian cells. Furthermore, we show that Red Broccoli can be fused to a S-adenosyl methionine (SAM)-binding aptamer to generate a red fluorescent RNA-based sensor that enables imaging of SAM in live mammalian cells. These results reveal a red fluorescent fluorogenic aptamer that functions in mammalian cells and that can be readily developed into red fluorescent RNA-based sensors.