XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification

作者全名："Pu, Qinli; Ye, Yuanyuan; Hu, Juan; Xie, Cong; Zhou, Xi; Yu, Hongyan; Liao, Fangli; Jiang, Song; Jiang, Linshan; Xie, Guoming; Chen, Weixian"

作者地址："[Pu, Qinli; Ye, Yuanyuan; Hu, Juan; Xie, Cong; Liao, Fangli; Jiang, Song; Jiang, Linshan; Chen, Weixian] Chongqing Med Univ, Dept Lab Med, Affiliated Hosp 2, Chongqing 400010, Peoples R China; [Zhou, Xi; Yu, Hongyan; Xie, Guoming] Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Chinese Minist Educ, Chongqing 400016, Peoples R China"

通信作者："Chen, WX (通讯作者)，Chongqing Med Univ, Dept Lab Med, Affiliated Hosp 2, Chongqing 400010, Peoples R China.; Xie, GM (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Clin Lab Diagnost, Chinese Minist Educ, Chongqing 400016, Peoples R China."

来源：TALANTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000863227600009

JCR分区：Q1

影响因子：5.6

年份：2023

卷号：252

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：N6-methyladenosine; XNA probe; CRISPR; Cas12a; Electrochemical biosensor; Fluorescence

摘要："N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/ Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modi-fications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypo -methylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis."

基金机构："National Natural Science Foundation of China, China [81873971]; China Postdoctoral Science Foundation, China [2022M710561]; Graduate Scientific Research and Innovation Project of Chongqing, China [CYB19162]; Science and Technology Commission Foundation of Chongqing, China [Cstc2016jcyA0264]"

基金资助正文："This work was financially supported by grants from the National Natural Science Foundation of China, China (No. 81873971) , the China Postdoctoral Science Foundation, China (No. 2022M710561) , the Graduate Scientific Research and Innovation Project of Chongqing, China (No. CYB19162) and the Science and Technology Commission Foundation of Chongqing, China (No. Cstc2016jcyA0264) ."