Light-up RNA Research Articles

Highly sensitive multiplex detection of microRNA using light-up RNA aptamers

Abstract MicroRNAs (miRNA) involved in various biological processes can serve as important biomarkers for early diagnosis and prognosis of various cancers. Quantitative reverse transcription PCR is the gold standard method to detect miRNAs in clinical settings, but it requires expensive equipment and complicated primer designs, thus hindering its use in the field. Herein, we developed a simple, cost-effective strategy for the multiplex detection of miRNA using light-up RNA aptamers as the key detection component. In principle, the presence of target miRNAs produces a large amount of light-up RNA aptamers such as Spinach and Mango aptamers through strand displacement amplification in combination with transcription amplification. Consequently, light-up RNA aptamers bind to their corresponding fluorogens and emit highly enhanced fluorescence signals at different wavelengths, which enables the multiplex detection of target miRNAs. With the proposed strategy, target miRNAs such as miR21 and miR141 were simultaneously determined with limits of detection of 0.955 fM and 0.195 fM, respectively. This assay was successfully applied for the quantification of miRNAs in different cell lines, demonstrating its practical utility in clinical diagnosis and prognosis.

Light-up RNA aptamer signaling-CRISPR-Cas13a-based mix-and-read assays for profiling viable pathogenic bacteria

Viable pathogenic bacteria cause serious human diseases via systemic infections and food poisoning. Herein, we constructed a light-up RNA aptamer signaling-CRISPR-Cas13a assay enabling mix-and-read detection of viable pathogenic bacteria. Directly targeting pathogen RNAs via CRISPR-Cas13a allows precisely discriminating viable bacteria from dead bacteria. We introduced a light-up RNA aptamer, Broccoli, serving as the substate of activated CRISPR-Cas13a to monitor the presence of pathogen RNAs, eliminating the need to use chemically labeled RNA substrate. Sequentially, the assay allows a reverse transcription-free, nucleic acid amplification-free, and label-free quantification of RNA targets and viable pathogenic bacteria. It could detect as low as 10 CFU of Bacillus cereus and precisely quantify viable bacteria with a content ranging from 0% to 100% in 105 CFU total bacteria. The quantification of viable bacteria allows more accurately estimating the ability of B. cereus to spoil food. The RNA assay promises its use in point-of-use detection of viable pathogens and biosafety control.

Activatable CRISPR Transcriptional Circuits Generate Functional RNA for mRNA Sensing and Silencing.

CRISPR-dCas9 systems that are precisely activated by cell-specific information facilitate the development of smart sensors or therapeutic strategies. We report the development of an activatable dCas9 transcriptional circuit that enables sensing and silencing of mRNA in living cells using hybridization-mediated structure switching for gRNA activation. The gRNA is designed with the spacer sequence blocked by a hairpin structure, and mRNA hybridization induces gRNA structure switching and activates the transcription of reporter RNA. An mRNA sensor developed using a light-up RNA reporter shows high sensitivity and fast-response imaging of survivin mRNA in cells under drug treatments and different cell lines. Furthermore, a feedback circuit is engineered by incorporating a small hairpin RNA in the reporter RNA, demonstrating a smart strategy for dynamic sensing and silencing of survivin with induced tumor cell apoptosis. This circuit illustrates a broadly applicable platform for the development of cell-specific sensing and therapeutic strategies.

Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer.

Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be compatible with advanced super-resolution techniques, mainly due to poor photophysical properties of their small-molecule fluorogens. Here, we describe a detailed protocol for fluorescence microscopy of mRNA in live bacteria using the recently reported fluorogenic silicon rhodamine binding aptamer (SiRA) featuring excellent photophysical properties. Notably, with SiRA, we demonstrated the first aptamer-based RNA visualization using super-resolution (STED) microscopy. This imaging method can be especially valuable for visualization of RNA in prokaryotes since the size of a bacterium is only a few times greater than the optical resolution of a conventional microscope.

Genetically encoded light-up RNA aptamers and their applications for imaging and biosensing.

Intracellular small ligands and biomacromolecules are playing crucial roles not only as executors but also as regulators. It is essential to develop tools to investigate their dynamics to interrogate their functions and reflect the cellular status. Light-up RNA aptamers are RNA sequences that can bind with their cognate nonfluorescent fluorogens and greatly activate their fluorescence. The emergence of genetically encoded light-up RNA aptamers has provided fascinating tools for studying intracellular small ligands and biomacromolecules owing to their high fluorescence activation degree and facile programmability. Here we review the burgeoning field of light-up RNA aptamers. We first briefly introduce light-up RNA aptamers with a focus on the photophysical properties of the fluorogens. Then design strategies of genetically encoded light-up RNA aptamer based sensors including turn-on, signal amplification and ratiometric rationales are emphasized.

SiRA: A Silicon Rhodamine-Binding Aptamer for Live-Cell Super-Resolution RNA Imaging.

Although genetically encoded light-up RNA aptamers have become promising tools for visualizing and tracking RNAs in living cells, aptamer/ligand pairs that emit in the far-red and near-infrared (NIR) regions are still rare. In this work, we developed a light-up RNA aptamer that binds silicon rhodamines (SiRs). SiRs are photostable, NIR-emitting fluorophores that change their open-closed equilibrium between the noncolored spirolactone and the fluorescent zwitterion in response to their environment. This property is responsible for their high cell permeability and fluorogenic behavior. Aptamers binding to SiR were in vitro selected from a combinatorial RNA library. Sequencing, bioinformatic analysis, truncation, and mutational studies revealed a 50-nucleotide minimal aptamer, SiRA, which binds with nanomolar affinity to the target SiR. In addition to silicon rhodamines, SiRA binds structurally related rhodamines and carborhodamines, making it a versatile tool spanning the far-red region of the spectrum. Photophysical characterization showed that SiRA is remarkably resistant to photobleaching and constitutes the brightest far-red light-up aptamer system known to date owing to its favorable features: a fluorescence quantum yield of 0.98 and an extinction coefficient of 86 000 M-1cm-1. Using the SiRA system, we visualized the expression of RNAs in bacteria in no-wash live-cell imaging experiments and also report stimulated emission depletion (STED) super-resolution microscopy images of aptamer-based, fluorescently labeled mRNA in live cells. This work represents, to our knowledge, the first application of the popular SiR dyes and of intramolecular spirocyclization as a means of background reduction in the field of aptamer-based RNA imaging. We anticipate a high potential for this novel RNA labeling tool to address biological questions.

A transcription aptasensor: amplified, label-free and culture-independent detection of foodborne pathogens via light-up RNA aptamers.

We report a transcription aptasensor by using a light-up RNA aptamer. It allows for sensitive, label-free and culture-free detection of intact foodborne pathogens, and no separation, purification or enrichment processes are involved.

A single promoter system co-expressing RNA sensor with fluorescent proteins for quantitative mRNA imaging in living tumor cells.

Genetically encoded light-up RNA aptamers afford a valuable platform for developing RNA sensors toward live cell imaging. However, quantitative imaging of intracellular RNAs remains a grand challenge. Here we reported a novel genetically encoded RNA sensor strategy using a plasmid that expresses a splittable fusion of the RNA sensor and the GFP mRNA in an individual transcript using a single promoter system. This splittable fusion design enables synchronous co-expression of the RNA sensor with GFP mRNA while alleviates the interference with correct folding of RNA aptamers due to intramolecular hybridization. This single-promoter system is applied to ratiometric imaging of survivin mRNA in tumor cells. The results reveal that the ratiometric images dynamically correlated with survivin mRNA concentrations and allow quantitative imaging of survivin mRNA in different tumor cells. The RNA sensor strategy may provide a new paradigm for developing a robust imaging platform for quantitative mRNA studies in living cells.

Isothermal folding of a light-up bio-orthogonal RNA origami nanoribbon

RNA presents intringuing roles in many cellular processes and its versatility underpins many different applications in synthetic biology. Nonetheless, RNA origami as a method for nanofabrication is not yet fully explored and the majority of RNA nanostructures are based on natural pre-folded RNA. Here we describe a biologically inert and uniquely addressable RNA origami scaffold that self-assembles into a nanoribbon by seven staple strands. An algorithm is applied to generate a synthetic De Bruijn scaffold sequence that is characterized by the lack of biologically active sites and repetitions larger than a predetermined design parameter. This RNA scaffold and the complementary staples fold in a physiologically compatible isothermal condition. In order to monitor the folding, we designed a new split Broccoli aptamer system. The aptamer is divided into two nonfunctional sequences each of which is integrated into the 5′ or 3′ end of two staple strands complementary to the RNA scaffold. Using fluorescence measurements and in-gel imaging, we demonstrate that once RNA origami assembly occurs, the split aptamer sequences are brought into close proximity forming the aptamer and turning on the fluorescence. This light-up ‘bio-orthogonal’ RNA origami provides a prototype that can have potential for in vivo origami applications.

Light-up RNA aptamer enabled label-free protein detection via a proximity induced transcription assay.

A novel proximity induced transcription assay for highly sensitive protein detection based on protein mediated ligation of a DNA template with the transcription of a light-up RNA aptamer for signal amplification has been developed.

Light-Up RNA Aptamers and Their Cognate Fluorogens: From Their Development to Their Applications.

An RNA-based fluorogenic module consists of a light-up RNA aptamer able to specifically interact with a fluorogen to form a fluorescent complex. Over the past decade, significant efforts have been devoted to the development of such modules, which now cover the whole visible spectrum, as well as to their engineering to serve in a wide range of applications. In this review, we summarize the different strategies used to develop each partner (the fluorogen and the light-up RNA aptamer) prior to giving an overview of their applications that range from live-cell RNA imaging to the set-up of high-throughput drug screening pipelines. We then conclude with a critical discussion on the current limitations of these modules and how combining in vitro selection with screening approaches may help develop even better molecules.

Genetically Encoded Fluorescent RNA Sensor for Ratiometric Imaging of MicroRNA in Living Tumor Cells.

Light-up RNA aptamers are valuable tools for fluorescence imaging of RNA in living cells and thus for elucidating RNA functions and dynamics. However, no light-up RNA sensor has been reported for imaging of microRNAs (miRs) in mammalian cells. We report a novel genetically encoded RNA sensor for fluorescent imaging of miRs in living tumor cells using a light-up RNA aptamer that binds to sulforhodamine and separates it from a conjugated contact quencher. On the basis of the structural switching mechanism for molecular beacon, we show that the RNA sensor activates high-contrast fluorescence from the sulforhodamine-quencher conjugate when its stem-loop responsive motif hybridizes with target miR. The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to miR target. We also realize the RNA sensor for dual-emission, ratiometric imaging by coexpression of RNA sensor with GFP, enabling quantitative studies of target miR in living cells. Our design may provide a new paradigm for developing robust, sensitive light-up RNA sensors for RNA imaging applications.

ISpinach: a fluorogenic RNA aptamer optimized for in vitro applications.

Using random mutagenesis and high throughput screening by microfluidic-assisted In Vitro Compartmentalization, we report the isolation of an order of magnitude times brighter mutants of the light-up RNA aptamers Spinach that are far less salt-sensitive and with a much higher thermal stability than the parent molecule. Further engineering gave iSpinach, a molecule with folding and fluorescence properties surpassing those of all currently known aptamer based on the fluorogenic co-factor 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). We illustrate the potential of iSpinach in a new sensitive and high throughput-compatible fluorogenic assay that measures co-transcriptionally the catalytic constant (kcat) of a model ribozyme.

In vitro assembly of cubic RNA-based scaffolds designed in silico.

The organization of biological materials into versatile three-dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. Here we report a strategy to design three-dimensional nanoscale scaffolds that can be self-assembled from RNA with precise control over their shape, size and composition. These cubic nanoscaffolds are only ~13 nm in diameter and are composed of short oligonucleotides making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly is verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes is also demonstrated by incorporation of a light-up fluorescent RNA aptamer that is optimally active only upon full RNA assembly. Moreover, we show the RNA nano-scaffolds can self-assemble in isothermal conditions (37°C) during in vitro transcription, which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications.