CRISPR/Cas13a-triggered entropy-driven amplification for colorimetric and fluorescent dual-mode detection of microRNA.

MicroRNAs (miRNAs) are crucial biomarkers for the early detection and monitoring of disease progression of chronic obstructive pulmonary disease (COPD). Herein, we have devised a method for detecting miRNA using a combination of colorimetric and graphene oxide-based fluorescent techniques. The target miRNA in our design could precisely activate the trans-cleavage activity of the CRISPR-Cas13a system. The activated Cas13a enzyme cuts the "rUrU" section in the P1 probe, generating a nicking site to induce entropy-driven amplification (EDA). One of the available EDA products has the capability to unfold the hairpin probe, thereby initiating the catalytic hairpin assembly, exposing the G-quadruplex structure, facilitating the subsequent color response. The fuel strand labeled with Cy3 successfully established a double-stranded DNA structure with DNA3, and consequently the Cy3 would not be quenched by graphene oxide (GO). The implementation of the dual-mode technique in this method yields greater benefits in terms of improving the precision and consistency of the miRNA measurements. The developed method has the capability to fluorescently measure miRNA-21 levels down to a concentration of 5.8 fM. In addition, the analysis of miRNA targets from clinical samples using this method demonstrates its promising utility in the fields of biomedical research of COPD.

A novel ultrasensitive ECL sensor for DNA detection based on nicking endonuclease-assisted target recycling amplification, rolling circle amplification and hemin/G-quadruplex.

In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form "Y" junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O8(2-) system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

Common functional polymorphism within miR-146a and miR-196a-2 as susceptibility loci for hepatocellular carcinoma: An updated meta-analysis.

BACKGROUND: Mutations or single nucleotide polymorphisms (SNPs) within the gene region of microRNAs play an important role for the development of hepatocellular carcinoma (HCC). Extensive studies have tried to investigate the susceptibility role of miR-146a rs2819164 and miR-196a-2 rs11614913. However, these results are still inconsistent and inconclusive. We undertook a meta-analysis containing primarily Asian studies to assess the associations of the two SNPs with HCC risk. METHODS: 19 studies including miR-146a (7170 cases and 9443 controls) and 15 studies including miR-196a-2 (6417 cases and 7627 controls) were used for meta-analysis. Odds ratios and 95% CI were calculated to assess the association in five different genetic models. RESULTS: For the rs2910164 polymorphism of miR-146a, significantly increased risks for HCC were observed when all studies were pooled under two models (CG vs CC: OR = 1.11, 95% CI = 1.02-1.21, P = 0.021; GG + CG vs CC: OR = 1.11, 95% CI = 1.01-1.22, P = 0.035). For the rs11614913 polymorphism of miR-196a-2, significant increased risks for HCC development were observed when all studies were pooled under four models (C vs T: OR = 1.14, 95% CI = 1.06-1.23, P = 0.001; CC vs TT: OR = 1.31, 95% CI = 1.12-1.53, P = 0.001; CC + TC vs TT: OR = 1.16, 95% CI = 1.03-1.31, P = 0.018; CC vs TC + TT: OR = 1.14, 95% CI = 1.00-1.30, P = 0.043). CONCLUSION: Our results show that the two common SNPs within the miRNAs were associated with modest increased risk of HCC (OR < 1.6), especially in the Asian population. Larger population-based studies validating these results are needed.