Development of a N6-methyladenosine-directed single quantum dot-based biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues

Ming-Hao Liu; Wan-Tong Yu; Ning-Ning Zhao; Jian-Ge Qiu; Bing-Hua Jiang; Yan Zhang; Chun-Yang Zhang

doi:10.1016/j.aca.2023.341796

Development of a N⁶-methyladenosine-directed single quantum dot-based biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues

Anal Chim Acta. 2023 Oct 23:1279:341796. doi: 10.1016/j.aca.2023.341796. Epub 2023 Sep 7.

Authors

Ming-Hao Liu¹, Wan-Tong Yu², Ning-Ning Zhao², Jian-Ge Qiu³, Bing-Hua Jiang⁴, Yan Zhang⁵, Chun-Yang Zhang⁶

Affiliations

¹ Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Zhengzhou University, Zhengzhou, 450052, China; College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China; College of Chemistry and Chemical Engineering, Qilu Normal University, Jinan, 250200, China.
² College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
³ Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Zhengzhou University, Zhengzhou, 450052, China.
⁴ Translational Medicine Center, The First Affiliated Hospital of Zhengzhou University, The Academy of Medical Sciences, Zhengzhou University, Zhengzhou, 450052, China. Electronic address: binghjiang@zzu.edu.cn.
⁵ College of Chemistry and Chemical Engineering, Qilu Normal University, Jinan, 250200, China. Electronic address: yanzhang_2003@163.com.
⁶ School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, China. Electronic address: zhangcy@seu.edu.cn.

PMID: 37827689
DOI: 10.1016/j.aca.2023.341796

Abstract

The METTL3/14 complex is an important RNA N⁶-Methyladenosine (m⁶A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m⁶A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10^-17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery.

Keywords: Breast cancer; Förster resonance energy transfer; METTL3/14 complex; Quantum dot; RNA m(6)A methyltransferase.

MeSH terms

Biosensing Techniques*
Breast Neoplasms* / diagnosis
DNA / chemistry
Female
Humans
Methyltransferases
Quantum Dots* / chemistry
RNA

Substances

cyanine dye 5
N-methyladenosine
DNA
Methyltransferases
RNA
METTL3 protein, human