5-Hydroxymethylcytosine Glucosylation-Triggered Helicase-Dependent Amplification-Based Fluorescent Biosensor for Sensitive Detection of beta-Glucosyltransferase with Zero Background Signal

Key Words:HYDROXYMETHYLATED DNA DETECTION; ELECTROCHEMICAL BIOSENSOR; ISOTHERMAL AMPLIFICATION; ULTRASENSITIVE DETECTION; ALKALINE-PHOSPHATASE; ASSAY; 5-METHYLCYTOSINE; METHYLATION; FAMILY; TET1

Abstract:beta-glucosyltransferase (beta-GT) catalyzes the glucosylation of 5-hydroxymethylcytosine (5-hmC) to enable the survival of bacteriophage and parasite in host cells, and it is a critical tool enzyme for 5-hmC assay. However, few methods are available for beta-glucosyltransferase assay, and they usually have the drawbacks of radioactive contamination, high background, laborious procedures, and unsatisfactory sensitivity. Herein, we develop a new fluorescent biosensor with zero background signal for sensitive detection of beta-GT activity based on 5-hmC glucosylation-triggered helicase-dependent amplification (HDA). The detection probe we designed may act as both a probe for beta-GT recognition and a template for HDA amplification. The beta-GT-catalyzed 5-hmC glucosylation can protect the detection probes from both the cleavage by MfeI restrictive enzyme and the digestion by exonucleases I and III. The remaining detection probes can subsequently act as the templates for exponential HDA amplification to generate numerous double-stranded DNA products, which can be easily detected by SYBR Green I in a label-free manner. The zero background can be achieved by efficient elimination of primer-dimer nonspecific amplification and complete digestion of nonglucosylated detection probes. This biosensor exhibits high sensitivity and good specificity, and it can be further used to analyze beta-GT kinetic parameters and screen the inhibitors, providing a powerful platform for deeper understanding of beta-GT biological functions and promoting beta-GT-related epigenetic studies. Furthermore, this biosensor can be extended to detect various DNA-modifying enzymes by simply replacing the recognition sequence and restriction enzyme.

Volume:92

Issue:24

Translation or Not:no