Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine.

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Small Molecule-Inducible and Photoactivatable Cellular RNA N1-Methyladenosine Editing.

N1-methyladenosine (m1A) modification is one of the most prevalent epigenetic modifications on RNA. Given the vital role of m1A modification in RNA processing such as splicing, stability and translation, developing a precise and controllable m1A editing tool is pivotal for in-depth investigating the biological functions of m1A. In this study, we developed an abscisic acid (ABA)-inducible and reversible m1A demethylation tool (termed AI-dm1A), which targets specific transcripts by combining the chemical proximity-induction techniques with the CRISPR/dCas13b system and ALKBH3. We successfully employed AI-dm1A to selectively demethylate the m1A modifications at A8422 of MALAT1 RNA, and this demethylation process could be reversed by removing ABA. Furthermore, we validated its demethylation function on various types of cellular RNAs including mRNA, rRNA and lncRNA. Additionally, we used AI-dm1A to specifically demethylate m1A on ATP5D mRNA, which promoted ATP5D expression and enhanced the glycolysis activity of tumor cells. Conversely, by replacing the demethylase ALKBH3 with methyltransferase TRMT61A, we also developed a controllable m1A methylation tool, namely AI-m1A. Finally, we caged ABA by 4,5-dimethoxy-2-nitrobenzyl (DMNB) to achieve light-inducible m1A methylation or demethylation on specific transcripts. Collectively, our m1A editing tool enables us to flexibly study how m1A modifications on specific transcript influence biological functions and phenotypes.

Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine.

N6-methyladenosine (m6A) has recently gained much attention due to its diverse biological functions. Currently, the commonly used detection methods for locus-specific m6A marks are complicated to operate, it is difficult to quantify the methylation level, and they have high false-positive levels. Here, we report a new method for locus-specific m6A detection based on the methylate-sensitive endonuclease activity of MazF and the simultaneous amplification and testing (SAT) method, termed "m6A-MazF-SAT". Mechanically, MazF fails to cleave the A (m6A) CA motif; therefore, the undigested template can be SAT-amplified using specific probes targeting the upstream and downstream of sites of interest. Fluorescent signals of SAT amplification can be detected by real-time PCR, and therefore, they achieve the detection of m6A existence. After the condition optimization, m6A-MazF-SAT can significantly, accurately, and rapidly detect the m6A-modified sites in mRNA, rRNA, and lncRNA at the fmol level, as well as 10% m6A at the fmol level. In addition, m6A-MazF-SAT can quantify the abundance of target m6A in biological samples and can be used for the inhibitor selection of m6A-related enzymes. Together, we offer a new approach to detect locus-specific m6A both qualitatively and quantitatively; it is easy to operate, results can be obtained rapidly, and it has low false-positive levels and high repeatability.

A Fenton-Like Nanocatalyst Based on Easily Separated Magnetic Nanorings for Oxidation and Degradation of Dye Pollutant.

In this study, uniform Fe3O4 magnetic nanorings (Fe3O4-MNRs) were prepared through a simple hydrothermal method. The morphology, magnetic properties, and structure of the product were characterized by transmission electron microscope (TEM), scanning electron microscope (SEM), high resolution transmission electron microscopy (HRTEM), vibrating sample magnetometer (VSM), X-ray powder diffraction (XRD), and X-ray photoelectron spectroscopy (XPS), respectively. The Fe3O4-MNRs were used as Fenton-like catalysts in the presence of hydrogen peroxide (H2O2) and showed excellent Fenton-catalytic activity for degradation of organic dyes such as Methylene blue (MB), Rhodamine B (RhB), and Bromophenol blue (BPB). Furthermore, the obtained Fe3O4-MNRs could be recycled after used for several times and still remained in a relative high activity and could rapidly be separated from the reaction medium using a magnet without considerable loss. All results reveal that Fe3O4-MNRs have potential for the treatment of dyes pollutants.