A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection

Human Sun; Shiying Zhou; Yin Liu; Peng Lu; Na Qi; Guixue Wang; Mei Yang; Danqun Huo; Changjun Hou

doi:10.1016/j.aca.2022.340732

A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection

Anal Chim Acta. 2023 Jan 25:1239:340732. doi: 10.1016/j.aca.2022.340732. Epub 2022 Dec 20.

Authors

Human Sun¹, Shiying Zhou², Yin Liu², Peng Lu³, Na Qi², Guixue Wang², Mei Yang⁴, Danqun Huo⁵, Changjun Hou⁶

Affiliations

¹ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, PR China. Electronic address: 398015979@qq.com.
² Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, PR China.
³ Chongqing University Three Gorges Hospital, Chongqing, 404000, PR China.
⁴ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, PR China. Electronic address: yangmei@cqu.edu.cn.
⁵ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China. Electronic address: huodq@cqu.edu.cn.
⁶ Key Laboratory for Biorheological Science and Technology of Ministry of Education, Bioengineering College, Chongqing University, Chongqing, 400044, PR China; National Facility for Translational Medicine, Shanghai Jiao Tong University, Shanghai, 200240, PR China. Electronic address: houcj@cqu.edu.cn.

PMID: 36628729
DOI: 10.1016/j.aca.2022.340732

Abstract

DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH₂-modified 3' end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10^-4 U/mL and a broad detection range from 2 × 10^-4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.

Keywords: CRISPR/Cas12a; DNA methyltransferase; Exponential amplification reaction; Fluorescence.

MeSH terms

Biosensing Techniques* / methods
CRISPR-Cas Systems
Coloring Agents
DNA / genetics
DNA / metabolism
DNA Methylation
DNA Modification Methylases / metabolism
Site-Specific DNA-Methyltransferase (Adenine-Specific)*

Substances

Site-Specific DNA-Methyltransferase (Adenine-Specific)
DNA Modification Methylases
Coloring Agents
DNA