The fluorescent biosensor for detecting N6 methyladenine FzD5 mRNA and MazF activity

N-6 methyladenine (m(6)A) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (CSDA) and CRISPR/Cas12a to detect m(6)A FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to m(6)A RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for m(6)A RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 x 10(-4) U mL(-1). More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting m(6)A FzD5 mRNA and MazF activity. (C) 2021 Elsevier B.V. All rights reserved. N6 methyladenine (m(6)A) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (CSDA) and CRISPR/Cas12a to detect m(6)A FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to m6A RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for m(6)A RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 x 10(-4) U mL(-1). More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting m(6)A FzD5 mRNA and MazF activity. (C) 2021 Elsevier B.V. All rights reserved.

Automated summary

An ultra-sensitive biosensor based on MazF, cascaded strand displacement amplification (CSDA) and CRISPR/Cas12a was developed for the detection of m(6)A FzD5 mRNA, showing excellent sensitivity and detection limit.