Exonuclease-assisted enrichment and base resolution analysis of pseudouridine in single-stranded RNA.

Pseudouridine (Ψ) is one of the most abundant RNA modifications, playing crucial roles in various biological processes. Identifying Ψ sites is vital for understanding their functions. In this study, we proposed a novel method for identifying Ψ sites with an improved signal-to-noise ratio.

Ultrasensitive Method Enables Liquid Biopsy for the Precise Detection of Circulating MicroRNAs.

Due to invasive and serial examinations of bioactive molecules, liquid biopsy (LB) has emerged as a rapid and reliable solution for early disease detection and monitoring. Developing portable devices with high specificity and sensitivity for LB is highly valuable. To realize a generalized approach to increase the sensitivity of LB, we developed an ultrasensitive diagnostic biochip based on the amplification of miRNA by recombinase polymerase amplification and the significant enhancement of fluorescence signals by photonic crystal (PC) materials.

RNA modifications identification based on chemical reactions.

RNA modification identification is an emerging field in epigenetics due to its indispensable regulatory role in the cell life cycle. With advancements in identification methods, an increasing number of RNA modifications has been discovered, thereby driving the development of more efficient and accurate techniques for localizing modified RNAs and elucidating their functions. High-throughput sequencing approaches for modified RNA detection can be categorized into antibody-based, enzymatic-based, and chemical-labeling-based methods.

A bisulfite-assisted and ligation-based qPCR amplification technology for locus-specific pseudouridine detection at base resolution.

Over 150 types of chemical modifications have been identified in RNA to date, with pseudouridine (Ψ) being one of the most prevalent modifications in RNA. Ψ plays vital roles in various biological processes, and precise, base-resolution detection methods are fundamental for deep analysis of its distribution and function. In this study, we introduced a novel base-resolution Ψ detection method named pseU-TRACE.

Steric hindrance of -methyl in mA and its application for specific loci detection.

We found that the methyl group of -methyladenine is able to hinder the methylation of -methyladenine at the position by DMS. Based on this, we have devised a novel method for detecting -methyladenine, which was successfully applied to identify specific mA loci in 28S rRNA.

Locus-specific detection of pseudouridine with CRISPR-Cas13a.

We combined the CRISPR-Cas13a system with CMC chemical labeling, developing an approach that enables precise identification of pseudouridine (Ψ) sites at specific loci within ribosomal RNA (rRNA), messenger RNA (mRNA) and small nuclear RNAs (snRNA). This method, with good efficiency and simplicity, detects Ψ sites through fluorescence measurement, providing a straightforward and fast validation for targeted Ψ sites of interest.

Method for the extraction of circulating nucleic acids based on MOF reveals cell-free RNA signatures in liver cancer.

Cell-free RNA (cfRNA) allows assessment of health, status, and phenotype of a variety of human organs and is a potential biomarker to non-invasively diagnose numerous diseases. Nevertheless, there is a lack of highly efficient and bias-free cfRNA isolation technologies due to the low abundance and instability of cfRNA. Here, we developed a reproducible and high-efficiency isolation technology for different types of cell-free nucleic acids (containing cfRNA and viral RNA) in serum/plasma based on the inclusion of nucleic acids by metal-organic framework (MOF) materials, which greatly improved the isolation efficiency and was able to preserve RNA integrity compared with the most widely used research kit method.

Fibroblastic reticular cell-derived exosomes are a promising therapeutic approach for septic acute kidney injury.

Sepsis-induced acute kidney injury (S-AKI) is highly lethal, and effective drugs for treatment are scarce. Previously, we reported the robust therapeutic efficacy of fibroblastic reticular cells (FRCs) in sepsis. Here, we demonstrate the ability of FRC-derived exosomes (FRC-Exos) to improve C57BL/6 mouse kidney function following cecal ligation and puncture-induced sepsis.

Antibody-Free Fluorine-Assisted Metabolic Sequencing of RNA -Acetylcytidine.

-Acetylcytidine (acC) has been found to affect a variety of cellular and biological processes. For a mechanistic understanding of the roles of acC in biology and disease, we present an antibody-free, fluorine-assisted metabolic sequencing method to detect RNA acC, called "FAM-seq". We successfully applied FAM-seq to profile acC landscapes in human 293T, HeLa, and MDA cell lines in parallel with the reported acRIP-seq method.

Detection, Clinical Application, and Manipulation of RNA Modifications.

ConspectusWith increasing research interest, more than 170 types of chemical modifications of RNA have been characterized. The epigenetic modifications of RNA do not alter the primary sequence of RNA but modulate the gene activity. Increasing numbers of regulatory functions of these RNA modifications, particularly in controlling mRNA fate and gene expression, are being discovered.

Transcriptome-wide profiling of A-to-I RNA editing by Slic-seq.

Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional processing event involved in diversifying the transcriptome and is responsible for various biological processes. In this context, we developed a new method based on the highly selective cleavage activity of Endonuclease V against Inosine and the universal activity of sodium periodate against all RNAs to enrich the inosine-containing RNA and accurately identify the editing sites. We validated the reliability of our method in human brain in both Alu and non-Alu elements.

Transcriptome-wide identification of single-stranded RNA binding proteins.

RNA-protein interactions are precisely regulated by RNA secondary structures in various biological processes. Large-scale identification of proteins that interact with particular RNA structure is important to the RBPome. Herein, a kethoxal assisted single-stranded RNA interactome capture (KASRIC) strategy was developed to globally identify single-stranded RNA binding proteins (ssRBPs).

A CRISPR-Cas and Tat Peptide with Fluorescent RNA Aptamer System for Signal Amplification in RNA Imaging.

We reported on an efficient RNA imaging strategy based on a CRISPR-Cas and Tat peptide with a fluorescent RNA aptamer (TRAP-tag). Using modified CRISPR-Cas RNA hairpin binding proteins fused with a Tat peptide array that recruits modified RNA aptamers, this simple and sensitive strategy is capable of visualizing endogenous RNA in cells with high precision and efficiency. In addition, the modular design of the CRISPR-TRAP-tag facilitates the substitution of sgRNAs, RNA hairpin binding proteins, and aptamers in order to optimize imaging quality and live cell affinity.

Mitochondria-targeting NIR AIEgens with cationic amphiphilic character for imaging and efficient photodynamic therapy.

A new dual-cationic amphiphilic AIEgen TPhBT-PyP with NIR emission and efficient O generation was designed. The amphiphilicity of TPhBT-PyP was tuned with dual-positive charges of pyridinium and TPP groups, efficiently targeting mitochondria and distinguishing Gram-positive bacteria. TPhBT-PyP performed well in photodynamic therapy, inducing cancer cell apoptosis and killing .

Live-cell RNA imaging using the CRISPR-dCas13 system with modified sgRNAs appended with fluorescent RNA aptamers.

The development of RNA imaging strategies in live cells is essential to improve our understanding of their role in various cellular functions. We report an efficient RNA imaging method based on the CRISPR-dPspCas13b system with fluorescent RNA aptamers in sgRNA (CasFAS) in live cells. Using modified sgRNA attached to fluorescent RNA aptamers that showed reduced background fluorescence, this approach provides a simple, sensitive way to image and track endogenous RNA with high accuracy and efficiency.

Transcriptome-wide profiling of -methyladenosine a selective chemical labeling method.

Studies of chemical modifications on RNA have ushered in the field of epitranscriptomics. -Methyladenosine (mA) is the most typical RNA modification and is indispensable for basic biological processes. This study presents a chemical pulldown method (mA-ORL-Seq) for transcriptome-wide profiling of mA.

Inert Pepper aptamer-mediated endogenous mRNA recognition and imaging in living cells.

The development of RNA aptamers/fluorophores system is highly desirable for understanding the dynamic molecular biology of RNAs in vivo. Peppers-based imaging systems have been reported and applied for mRNA imaging in living cells. However, the need to insert corresponding RNA aptamer sequences into target RNAs and relatively low fluorescence signal limit its application in endogenous mRNA imaging.

KAS-seq: genome-wide sequencing of single-stranded DNA by N-kethoxal-assisted labeling.

Transcription and its dynamics are crucial for gene expression regulation. However, very few methods can directly read out transcriptional activity with low-input material and high temporal resolution. This protocol describes KAS-seq, a robust and sensitive approach for capturing genome-wide single-stranded DNA (ssDNA) profiles using N-kethoxal-assisted labeling.

Intelligent demethylase-driven DNAzyme sensor for highly reliable metal-ion imaging in living cells.

The accurate intracellular imaging of metal ions requires an exquisite site-specific activation of metal-ion sensors, for which the pervasive epigenetic regulation strategy can serve as an ideal alternative thanks to its orthogonal control feature and endogenous cell/tissue-specific expression pattern. Herein, a simple yet versatile demethylation strategy was proposed for on-site repairing-to-activating the metal-ion-targeting DNAzyme and for achieving the accurate site-specific imaging of metal ions in live cells. This endogenous epigenetic demethylation-regulating DNAzyme system was prepared by modifying the DNAzyme with an mA methylation group that incapacitates the DNAzyme probe, thus eliminating possible off-site signal leakage, while the cell-specific demethylase-mediated removal of methylation modification could efficiently restore the initial catalytic DNAzyme for sensing metal ions, thus allowing a high-contrast bioimaging in live cells.

Single-Base Resolution Mapping Reveals Distinct 5-Formylcytidine in mRNAs.

5-Formylcytidine (fC) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria and essential for mitochondrial protein synthesis. Here, we show a method to detect fC modifications in RNA and a transcriptome-wide fC mapping technique, named fC-seq. It is developed based on the treatment of pyridine borane, which can reduce fC to 5,6-dihydrouracil, thus inducing C-to-T transition in fC sites during PCR to achieve single-base resolution detection.

A longitudinal sampling study of transcriptomic and epigenetic profiles in patients with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease caused by a new type of SFTS virus (SFTSV). Here, a longitudinal sampling study is conducted to explore the differences in transcript levels after SFTSV infection, and to characterize the transcriptomic and epigenetic profiles of hospitalized patients. The results reveal significant changes in the mRNA expression of certain genes from onset to recovery.

One-pot fluorescent assay for sensitive detection of APOBEC3A activity.

We reported a one-pot fluorescence-based assay to quantitively detect A3A activity combined with cytosine deamination and uracil excision. After deamination by A3A and USER enzyme treatment, the fluorescent turn-on effect at 520 nm was observed, which can be used to evaluate the A3A activity and screen inhibitors.

Highly sensitive detection of 6mA at single-base resolution based on A-C mismatch.

We first demonstrated that 6mA can be selectively recognized based on the selective ligation reaction of DNA ligase toward A-C mismatch and 6mA-C mismatch. This method, when further combined with amplification using RCA, achieved highly sensitive identification of 6mA in dsDNA at single-base resolution.