A fluorescence strategy for circRNA quantification in tumor cells based on T7 nuclease-assisted cycling enzymatic amplification

Hongke Qu; Mingjian Chen; Junshang Ge; Xiangyan Zhang; Shuyi He; Fang Xiong; Qijia Yan; Shanshan Zhang; Zhaojian Gong; Can Guo; Fuyan Wang; Zhaoyang Zeng; Xiaoling Li; Guiyuan Li; Wei Xiong; Xu Wu

doi:10.1016/j.aca.2021.339210

A fluorescence strategy for circRNA quantification in tumor cells based on T7 nuclease-assisted cycling enzymatic amplification

Anal Chim Acta. 2022 Jan 2:1189:339210. doi: 10.1016/j.aca.2021.339210. Epub 2021 Oct 26.

Authors

Hongke Qu¹, Mingjian Chen², Junshang Ge², Xiangyan Zhang³, Shuyi He², Fang Xiong⁴, Qijia Yan⁴, Shanshan Zhang⁴, Zhaojian Gong⁵, Can Guo¹, Fuyan Wang¹, Zhaoyang Zeng⁶, Xiaoling Li⁶, Guiyuan Li⁶, Wei Xiong⁷, Xu Wu⁸

Affiliations

¹ NHC Key Laboratory of Carcinogenesis and Human Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China; Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute and School of Basic Medicine Sciences, Central South University, Changsha, Hunan, China.
² Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute and School of Basic Medicine Sciences, Central South University, Changsha, Hunan, China.
³ Department of Forensic Science, School of Basic Medical Sciences, Central South University, Changsha, Hunan, China.
⁴ Department of Stomatology, Xiangya Hospital, Central South University, Changsha, Hunan, China.
⁵ Department of Oral and Maxillofacial Surgery, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China.
⁶ NHC Key Laboratory of Carcinogenesis and Human Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China; Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute and School of Basic Medicine Sciences, Central South University, Changsha, Hunan, China; Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome Research Center, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China.
⁷ NHC Key Laboratory of Carcinogenesis and Human Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China; Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute and School of Basic Medicine Sciences, Central South University, Changsha, Hunan, China; Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome Research Center, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China. Electronic address: xiongwei@csu.edu.cn.
⁸ NHC Key Laboratory of Carcinogenesis and Human Key Laboratory of Cancer Metabolism, Hunan Cancer Hospital and the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan, China; Key Laboratory of Carcinogenesis and Cancer Invasion of the Chinese Ministry of Education, Cancer Research Institute and School of Basic Medicine Sciences, Central South University, Changsha, Hunan, China; Hunan Key Laboratory of Nonresolving Inflammation and Cancer, Disease Genome Research Center, The Third Xiangya Hospital, Central South University, Changsha, Hunan, China. Electronic address: wuxu1028@csu.edu.cn.

PMID: 34815051
DOI: 10.1016/j.aca.2021.339210

Abstract

Circular Ribonucleic Acid (CircRNA) plays regulatory roles in many biological processes, such as tumors and metabolic diseases. Due to the fact that circRNA is more stable and conservative than linear RNA, circRNA has become a potential biomarker in early clinical diagnosis and biomedical research. Therefore, the quantification of circRNA expression level is of importance for understanding their functions and their applications for disease diagnosis and treatment. Nevertheless, due to the low abundance of circRNA, it is still a challenge for the analysis of circRNA in cells. Herein, we proposed a sensitive detection method for circRNA based on the T7 exonuclease-assisted cycling enzymatic amplification. The fluorescent sensor was constructed by a hairpin molecular beacon and T7 exonuclease. With the cycling enzymatic amplification process, this sensor achieved the limit of detection of 1 pM with a good linear correlation in the range of 0-100 pM (R² = 0.9891) using circBART2.2 as a model. Furthermore, we applied the proposed method in the determination of circBART2.2 in cell lysates. The results demonstrated that this method has promising applications in early diagnosis of Epstein-Barr virus (EBV) infection-related diseases using circRNA as the biomarker.

Keywords: CircBART2.2; CircRNA; Enzyme-assisted cycling amplification; Fluorescent sensor; Molecular beacon.

MeSH terms

Cell Count
Epstein-Barr Virus Infections*
Herpesvirus 4, Human
Humans
Limit of Detection
RNA, Circular*
Spectrometry, Fluorescence

Substances

RNA, Circular