5-Hydroxymethylcytosine Glucosylation-Triggered Helicase-Dependent Amplification-Based Fluorescent Biosensor for Sensitive Detection of β-Glucosyltransferase with Zero Background Signal.
Meng LiuChen-Chen LiXi-Liang LuoFei MaShuangshuang ZhangPublished in: Analytical chemistry (2020)
β-glucosyltransferase (β-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 β-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 β-GT activity based on 5-hmC glucosylation-triggered helicase-dependent amplification (HDA). The detection probe we designed may act as both a probe for β-GT recognition and a template for HDA amplification. The β-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 β-GT kinetic parameters and screen the inhibitors, providing a powerful platform for deeper understanding of β-GT biological functions and promoting β-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.
Keyphrases
- label free
- sensitive detection
- nucleic acid
- quantum dots
- living cells
- loop mediated isothermal amplification
- single molecule
- high throughput
- small molecule
- circulating tumor
- gene expression
- fluorescence imaging
- histone deacetylase
- risk assessment
- cell death
- high resolution
- drinking water
- cell cycle arrest
- signaling pathway
- oxidative stress
- photodynamic therapy
- single cell
- heavy metals
- toxoplasma gondii
- mass spectrometry
- real time pcr
- free survival
- gold nanoparticles