Here, we report that m6A significantly hinders DNA- and RNA-directed DNA synthesis, and a quantitative analysis of m6A in RNA or DNA context has been achieved..

Developing portable and sensitive devices for point of care detection of low abundance bioactive molecules is highly valuable in early diagnosis of disease. Herein, an ultrasensitive photonic crystals-assisted rolling circle amplification (PCs-RCA) biochip was constructed and further applied to circulating microRNAs (miRNAs) detection in serum. The biochip integrated the optical signal enhancement capability of biomimetic PCs surface with the thousand-fold signal amplification feature of RCA. The biomimetic PCs displayed periodic dielectric nanostructure and significantly enhanced the signal intensity of RCA reaction, leading to efficient improvement of detection sensitivity. A limit of detection (LOD) as low as 0.7 aM was obtained on the PCs-RCA biochip, and the LOD was 7 orders of magnitude lower than that of standard RCA. Moreover, the PCs-RCA biochip could discriminate a single base variation in miRNAs. Accurate quantification of ultralow-abundance circulating miRNAs in clinical serum samples was further achieved with the PCs-RCA biochip, and patients with the nonsmall cell lung carcinoma were successfully distinguished from healthy donors. The PCs-RCA biochip can detect bioactive molecules with ultrahigh sensitivity and good specificity, making it valuable in clinical disease diagnosis and health assessment.

G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.

The analysis of DNA methylation and MTase (methyltransferase) activity is important in epigenetic study. We have developed a novel strategy for sensitive analysis of MTase activity based on a hairpin shaped DNAzyme; 8-17 DNAzyme amplicon has been adopted and found to be very effective in such analysis.