Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA.

MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.

Gold Nanoflares with Computing Function as Smart Diagnostic Automata for Multi-miRNA Patterns in Living Cells.

Investigating the multimolecule patterns in living cells is of vital importance for clinical and biomedical studies. Herein, we reported for the first time the engineering of gold nanoflares as smart automata to implement computing-based diagnosis in living mammalian cells. Defining the logic combinations of miR122 and miR21 as the detection patterns, the corresponding OR and AND diagnostic automata were designed. The results showed that they could recognize the correct patterns rapidly and sensitively. The automata could enter cells via self-delivery and have good biocompatibility. They enabled accurate diagnosis on miRNA signatures in different cell lines and differentiation of fluctuations in the same cell line at single cell resolution. Moreover, the automata afforded an innovative diagnostic mode. It simplified the complicated process of detecting, data-collecting, computing, and evaluating. The direct diagnosing result ("1" or "0") was exported according to the embedded computation code. It highlighted the new possibility of using smart automata for intelligent diagnostics and cancer therapy at single cell resolution.

Cascade Circuits on Self-Assembled DNA Polymers for Targeted RNA Imaging In Vivo.

DNA molecular probes have emerged as a powerful tool for RNA imaging. Hurdles in cell-specific delivery and other issues such as insufficient stability, limited sensitivity, or slow reaction kinetics, however, hinder the further application of DNA molecular probes in vivo. Herein, we report an aptamer-tethered DNA polymer for cell-specific transportation and amplified imaging of RNA in vivo via a DNA cascade reaction. DNA polymers are constructed through an initiator-triggered hybridization chain reaction using two functional DNA monomers. The prepared DNA polymers show low cytotoxicity and good stability against nuclease degradation and enable cell-specific transportation of DNA circuits via aptamer-receptor binding. Moreover, assembling the reactants of hairpins C1 and C2 on the DNA polymers accelerates the response kinetics and improves the sensitivity of the cascade reaction. We also show that the DNA polymers enable efficient imaging of microRNA-21 in live cells and in vivo via intravenous injection. The DNA polymers provide a valuable platform for targeted and amplified RNA imaging in vivo, which holds great implications for early clinical diagnosis and therapy.

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.

Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH.

DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on a cell's surface and undergoes pH-responsive triplex-duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. Results reveal that the DNA nanomachine can be stably anchored on the cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility, and quantitative ability for extracellular pH detection and imaging. This cell surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

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.

Rapid one-pot isothermal amplification reassembled of fluorescent RNA aptamer for SARS-CoV-2 detection.

The outbreak of SARS-CoV-2 poses a serious threat to human life and health. A rapid nucleic acid tests can effectively curb the spread of the disease. With the advantages of fluorescent RNA aptamers, low background and high sensitivity. A variety of fluorescent RNA aptamer sensors have been developed for the detection of nucleic acid. Here, we report a hypersensitive detection platform in which SARS-CoV-2 initiates RTF-EXPAR to amplify trigger fragments. This activation leads to the reassembled of the SRB2 fluorescent RNA aptamer, restoring its secondary structure for SR-DN binding and turn-on fluorescence. The platform completes the assay in 30 min and all reactions occur in one tube. The detection limit is as low as 116 aM. Significantly, the platform's quantitative analyses were almost identical to qPCR results in simulated tests of positive samples. In conclusion, the platform is sensitive, accurate and provides a new protocol for point-of-care testing of viruses.

Small-Molecule-Mediated Split-Aptamer Assembly for Inducible CRISPR-dCas9 Transcription Activation.

Inducible CRISPR-dCas9 transcription system has become a powerful tool for transcription regulation and sensing. Here, we develop a new concept of small-molecule-mediated split-aptamer assembly for inducible CRISPR-dCas9 transcription activation, allowing quantitative detection and imaging of S-adenosyl methionine (SAM) in live cells. This inducible transcription system is designed by integrating one fragment of a split SAM aptamer to guide RNA (gRNA) and the other to MS2 arrays. SAM-mediated reassembly of the split fragments recruits an MCP-fused transcription activator to the gRNA-dCas9 complex, activating the expression of a near-infrared fluorescent protein for imaging. We demonstrate that this inducible transcription system achieves quantitative detection of SAM with high sensitivity in live cells. Our system shows that methionine adenosyltransferase 1A (MAT1A) and MAT2A can both catalyze SAM production in live cells and the SAM levels in cancer cells can be increased via upregulation of MAT1A mRNA by epigenetic inhibitors. This split-aptamer assembly strategy could afford a new approach for controlling the CRISPR-dCas9 system, enabling conditional transcription regulation in response to endogenous metabolites in live cells.

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.

Spinach-based fluorescent light-up biosensors for multiplexed and label-free detection of microRNAs.

A novel Spinach-based fluorescent light-up biosensor utilizing the T7 in vitro transcription process to generate unmodified Spinach sequences for multiplexed microRNA detection has been developed.

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.