Light-up RNA Research Articles

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

Label-Free and DNAzyme-Mediated Biosensor with a High Signal-to-Noise Ratio for a Lumpy Skin Disease Virus Assay.

Lumpy skin disease virus (LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV. The method integrated recombinase polymerase amplification (RPA), CRISPR/Cas12a, 10-23 DNAzyme, and Baby Spinach RNA aptamer for triple cascade signal amplification. Based on highly sensitive and specific RPA and CRISPR/Cas12a, DNAzyme achieved a third signal amplification. Additionally, the Baby Spinach RNA aptamer had stronger fluorescence signals and higher quantum yields. The label-free method had ultrahigh sensitivity with the actual detection limit as 1.29 copies·μL-1. The method was 100-fold more sensitive compared to RPA with Cas12a. Moreover, it had no cross-reactivity with viruses belonging to the Capripoxvirus, such as sheep pox virus and goat pox virus with genetic homology as 97%. Furthermore, the method displayed 100% accuracy in 50 actual samples. Therefore, the method based on RPA, Cas12a, and 10-23 DNAzyme had advantages in LSDV detection and provided a new solution for LSD prevention and control.

Fluorogenic RNA Aptamer-Based Amplification and Transcription Strategy for Label-free Sensing of Methyltransferase Activity in Complex Matrixes.

DNA methyltransferase is significant in cellular activities and gene expression, and its aberrant expression is closely linked to various cancers during initiation and progression. Currently, there is a great demand for reliable and label-free techniques for DNA methyltransferase evaluation in tumor diagnosis and cancer therapy. Herein, a low-background fluorescent RNA aptamer-based sensing approach for label-free quantification of cytosine-guanine (CpG) dinucleotides methyltransferase (M.SssI) is reported. The fluorogenic light-up RNA aptamers-based strategy exhibits high selectivity via restriction endonuclease, padlock-based recognition, and RNA transcription. By combining rolling circle amplification (RCA), and RNA transcription with fluorescence response of RNA aptamers of Spinach-dye compound, the proposed platform exhibited efficiently ultrahigh sensitivity toward M.SssI. Eventually, the detection can be achieved in a linear range of 0.02-100 U mL-1 with a detection limit of 1.6 × 10-3 U mL-1. Owing to these superior features, the method is further applied in serum samples spiked M.SssI, which delivers a recovery ranging from 92.0 to 107.0% and a relative standard deviation <7.0%, providing a promising and practical tool for determining M.SssI in complex biological matrices.

Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets.

The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor's performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Modular Approach for Rapid Identification of RNA-Based Sensors.

Detection of metabolites in real time and in whole cells requires effective molecular sensors. In this regard, fluorogenic light-up RNAs have recently become important tools for small-molecule detection in cells. However, the construction of light-up RNA sensors is an arduous task that requires structural knowledge of both the sensor and reporter RNA. De novo strategies for selecting sensors from RNA libraries are limited and are mostly restricted to known aptamers and riboswitches. Here, we provide a solution to this problem by developing a capture-SELEX variant that allows the obtained libraries and aptamers to be linked to fluorogenic RNAs in a modular and allosteric manner. The approach is generally applicable and allows for rapid modular allosteric assembly with green- or red-shifted fluorogenic RNAs.

Tetrahedral DNA nanostructures enhance transcription isothermal amplification for multiplex detection of non-coding RNAs

This study introduces an innovative detection system for multiple cancer biomarkers, employing transcription isothermal amplification methods in conjunction with a tetrahedral DNA nanostructure (TDN). We demonstrate that TDN enhances various transcription isothermal amplification methods by placing DNA probes in proximity. Notably, the TDN-enhanced split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR) system stands out for its optimal performance and operational simplicity, especially in identifying non-coding RNAs such as microRNAs and long non-coding RNAs (lncRNAs). Multiplex detection of lncRNAs was also achieved by generating distinct light-up RNA aptamers, each emitting unique fluorescence signals. The system effectively identified the target lncRNAs, demonstrating high sensitivity and selectivity in both cell lines and clinical samples. The system, utilizing the single enzyme T7 RNA polymerase, can be easily tailored for alternative targets by substituting target-specific sequences in DNA probes and seamlessly integrated with other isothermal amplification methods for greater sensitivity and accuracy in the detection of multiple cancer biomarkers.

Primer exchange reaction-coupled transcription isothermal amplification as a sensitive biomolecular assay.

We devised a novel strategy that relies on a combination of the primer exchange reaction (PER) with transcription isothermal amplification, termed PER-Trap, for a sensitive biomolecular assay. Its design allowed light-up RNA aptamers to be produced as the final product, leading to the generation of an amplified fluorescence signal. The utility of PER-Trap was successfully demonstrated by the detection of exosomes.

Direct fluoride monitoring using a fluorogenic RNA-based biosensor.

Fluorinated compounds, whether naturally occurring or from anthropogenic origin, have been extensively exploited in the last century. Degradation of these compounds by physical or biochemical processes is expected to result in the release of fluoride. Several fluoride detection mechanisms have been previously described. However, most of these methods are not compatible with high- and ultrahigh-throughput screening technologies, lack the ability to real-time monitor the increase of fluoride concentration in solution, or rely on costly reagents (such as cell-free expression systems). Our group recently developed "FluorMango" as the first completely RNA-based and direct fluoride-specific fluorogenic biosensor. To do so, we merged and engineered the Mango-III light-up RNA aptamer and the fluoride-specific aptamer derived from a riboswitch, crcB. In this chapter, we explain how this RNA-based biosensor can be produced in large scale before providing examples of how it can be used to quantitatively detect (end-point measurement) or monitor in real-time fluoride release in complex biological systems by translating it into measurable fluorescent signal.

Demethylation-activated light-up dual-color RNA aptamersensor for label-free detection of multiple demethylases in lung tissues

Methylation is one of the most prevalent epigenetic modifications in natural organisms, and the processes of methylation and demethylation are closely associated with cell growth, differentiation, gene transcription and expression. Abnormal methylation may lead to various human diseases including cancers. Simultaneous analysis of multiple DNA demethylases remains a huge challenge due to the requirement of diverse substrate probes and scarcity of proper signal transduction strategies. Herein, we propose a sensitive and label-free method for simultaneous monitoring of multiple DNA demethylases on the basis of demethylation-activated light-up dual-color RNA aptamers. The presence of targets AlkB homologue-3 (ALKBH3) and fat mass and obesity-associated enzyme (FTO) erases the methyl group in DNA substrate probes, activating the ligation-mediate bidirectional transcription amplification reaction to produce enormous Spinach and Mango aptamers. The resulting RNA aptamers (i.e., Spinach and Mango aptamers) can bind with their cognate nonfluorescent fluorogens (DFHBI and TO1-biotin) to significantly improve the fluorescence signals. This aptamersensor shows high specificity and sensitivity with a limit of detection (LOD) of 8.50 × 10−14 M for ALKBH3 and 6.80 × 10−14 M for FTO, and it can apply to screen DNA demethylase inhibitors, evaluate DNA demethylase kinetic parameters, and simultaneously measure multiple endogenous DNA demethylases in a single cell. Importantly, this aptamersensor can accurately discriminate the expressions of ALKBH3 and FTO between healthy tissues and non-small cell lung cancer (NSCLC) patient tissues, offering a powerful platform for clinical diagnosis and drug discovery.

Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

Real-time monitoring strategies for optimization of in vitro transcription and quality control of RNA.

RNA-based therapeutics and vaccines are opening up new avenues for modern medicine. To produce these useful RNA-based reagents, in vitro transcription (IVT) is an important reaction that primarily determines the yield and quality of the product. Therefore, IVT condition should be well optimized to achieve high yield and purity of transcribed RNAs. To this end, real-time monitoring of RNA production during IVT, which allows for fine tuning of the condition, would be required. Currently, light-up RNA aptamer and fluorescent dye pairs are considered as useful strategies to monitor IVT in real time. Fluorophore-labeled antisense probe-based methods can also be used for real-time IVT monitoring. In addition, a high-performance liquid chromatography (HPLC)-based method that can monitor IVT reagent consumption has been developed as a powerful tool to monitor IVT reaction in near real-time. This mini-review briefly introduces some strategies and examples for real-time IVT monitoring and discusses pros and cons of IVT monitoring methods.

Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA.

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.

Wash-Free and High-Contrast Imaging of Single-Nucleotide Variation in Single Cells Using Transcription-Amplified Light-Up RNA Aptamer.

Single-nucleotide variation (SNV) imaging can indicate cellular heterogeneity and spatial pattern, but it remains challenging to produce high-gain signal while also yielding single-nucleotide resolution. Herein, we developed a light-up strategy for visualizing SNVs based on transcription amplification, enabling wash-free and high-contrast imaging of SNVs inside cells. The discrimination of SNVs is achieved by ligase-assisted transcription reaction. Employing a light-up RNA aptamer as a reporter eliminates nonspecific probe binding and the washing process and contributes to a 2-fold improvement of signal gain compared to that using the fluorescence in situ hybridization (FISH) method. The method allowed us to precisely quantify drug-resistant strains in the bacteria mixture and identify drug-resistant Salmonella enterica (S. enterica) isolated from poultry farm. Using this approach, we explored the colonization features of drug-resistant and drug-sensitive S. enterica in the mice intestinal tract and screened the prebiotics for Salmonella colonization inhibition. The SNV imaging method promises for the interrogation of genotypes in physiological and pathological states at the single-cell level.

Switchable Fluorescent Light-Up Aptamers Based on Riboswitch Architectures.

Fluorescent light-up RNA aptamers (FLAPs) such as Spinach or Mango can bind small fluorogens and activate their fluorescence. Here, we adopt a switching mechanism otherwise found in riboswitches and use it to engineer switchable FLAPs that can be activated or repressed by trigger oligonucleotides or small metabolites. The fluorophore binding pocket of the FLAPs comprises guanine (G) quadruplexes, whose critical nucleotides can be sequestered by corresponding anti-FLAP sequences, leading to an inactive conformation and thus preventing association with the fluorophore. We modified the FLAPs with designed toehold hairpins that carry either an anti-FLAP or an anti-anti-FLAP sequence within the loop region. The addition of an input RNA molecule triggers a toehold-mediated strand invasion process that refolds the FLAP into an active or inactive configuration. Several of our designs display close-to-zero leak signals and correspondingly high ON/OFF fluorescence ratios. We also modified purine aptamers to sequester a partial anti-FLAP or an anti-anti-FLAP sequence to control the formation of the fluorogen-binding conformation, resulting in FLAPs whose fluorescence is activated or deactivated in the presence of guanine or adenine. We demonstrate that switching modules can be easily combined to generate FLAPs whose fluorescence depends on several inputs with different types of input logic.

Accelerating Cell-Free Biosensors by Entropy-Driven Assembly of Transcription Templates.

Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues.

Circular RNAs (circRNAs) as endogenous non-coding RNAs are characterized by covalently closed circular structures, and they widely exist in mammalian cells. The aberrant expression of circRNAs may result in various diseases. Herein, we demonstrate the construction of genetically encoded light-up RNA aptamers for ultrasensitive and label-free detection of circRNA mitochondrial tRNA translation optimization 1 (circMTO1) in cancer cells and tissues. The light-up RNA aptamers are generated by proximity ligation-activated recombinase polymerase amplification (RPA)-assisted transcription amplification. When circMTO1 is present, it initiates the proximity ligation reaction, activating RPA to produce numerous long double-stranded DNAs containing T7 promoters. Subsequently, the RPA products are identified by T7 RNA polymerase, initiating the transcription amplification reaction to generate abundant Spinach RNA aptamers. Spinach RNA aptamers can bind with DFHBI (3,5-difluoro-4-hydroxybenzylidene imidazolidinone) dye to produce a distinct fluorescence signal with near-zero background. This biosensor exhibits excellent selectivity and high sensitivity with a limit of detection of 2.54 aM. It can accurately monitor cellular circMTO1 at the single-cell level and discriminate the expression of circMTO1 between breast cancer patient tissues and healthy tissues. Notably, this biosensor can be employed to measure other nucleic acids by altering the corresponding target recognition sequences, providing a valuable platform for cancer diagnosis and biomedical study.

Biodegradable metal organic framework-mediated DNAzyme/light-up RNA aptamer transcription amplifications for label-free and highly sensitive sensing of telomerase in live cells

Sensitive and specific sensing of intracellular telomerase is of significance for early cancer diagnosis and treatment. And, efficient signal amplification technology is a powerful tool to achieve low level biomarker detection. However, the delivery and release of amplification reagents in live cells is currently a major challenge to achieve sensitive intracellular detection of biomarkers. Here, we describe the development of a powerful in-cell amplification approach for label-free and highly sensitive detection of telomerase in live cells via zeolitic imidazolate framework-8 (ZIF-8)-mediated DNAzyme/light-up RNA aptamer transcription amplification cascades. The amplification reagents can be readily transported into and released in the target cells by ZIF-8 under intracellular acidic environment. Telomerase then activates DNAzyme to trigger the DNAzyme-aided cleavage reaction and T7 RNA polymerase-initiated light-up RNA aptamer transcription cycles for the yield of many light-up RNA aptamers, which associate with the non-fluorescent dye to recover significant fluorescence for achieving intracellular telomerase imaging with high sensitivity in a label-free approach. Moreover, the proposed strategy can discriminate telomerase level variations in different cells and shows promising potentials for anticancer drug screening, which makes such method a robust bioassay for early tumor-related diagnosis and therapeutic monitoring.

Multipurpose advanced split T7 promoter-based transcription amplification for ultrasensitive molecular diagnostics

An accurate, convenient, and rapid diagnostic platform, which can be applied in facility-limited or point-of-care (POC) settings, is essential to help prevent the spread of infectious diseases and enable the most effective treatment to be selected. In this study, we describe the development of a new isothermal molecular diagnostic system named multipurpose advanced split T7 promoter-based transcription amplification (MASTER) for the rapid and ultrasensitive detection of various pathogens containing single-stranded RNA and double-stranded DNA. MASTER produces a large number of RNA amplicons in the presence of target pathogens, which generate fluorescence or colorimetric signals based on light-up RNA aptamers or lateral flow assays. Implementing MASTER at 37 °C for<1 h achieved the detection of a single copy per reaction without cross-reactivity. Moreover, the testing of 40 clinical samples revealed that MASTER exhibited excellent accuracy with 100% sensitivity and specificity for SARS-CoV-2 diagnosis. Furthermore, a one-pot MASTER system capable of accelerating practical applications was demonstrated, indicating that the MASTER system is a promising platform for the effective surveillance of various pathogens.

Dye disaggregation light-up aptamer for super-resolution RNA imaging

Genetically encoded light-up RNA aptamers have become powerful tools for visualizing RNAs in cells. However, fluorogenic dye/aptamer pairs used for super-resolution RNA imaging are still rare. Herein, we reported an environment-sensitive dye Nap which can light up its target RNA aptamer NapRA for aptamer-based RNA imaging with confocal and structured illumination microscopy (SIM). Nap was designed to be amphiphilic to assemble into nano-particles in water associated with the quenched fluorescence by the aggregation effects. NapRA was obtained from SELEX technology with a 12-round Nap-based selection and has a high binding affinity to Nap of Kd = 3.142 nM. The super affinity disintegrated the dye aggregate and resulted in a turn-on fluorescent signal. The light-up feature combined with high photostability made Nap/NapRA a useful system capable of super-resolution imaging of the expression of RNA in bacteria. This work represents the first application of dye aggregation-disaggregation for aptamer-based RNA imaging.