A Ligation-Based CRISPR-Cas Biosensing Platform for Non-Nucleic-Acid Targets Detection

• 1、College of Chemistry and Chemical Engineering, Hunan University

Abstract：With the discovery of the collateral cleavage activity of Class II types V and VI Cas proteins, the high-efficiency signal amplification capability of the CRISPR-Cas system has received widespread attention in the field of biosensing. However, the utility of current CRISPR-Cas biosensing system is mainly limited to nucleic acid molecular diagnosis, and its application in the detection of protein and small molecule has yet to be developed. Given that the DNA ligation catalyzed by DNA ligase relies on adenosine triphosphate (ATP) or nicotinamide adenine dinucleotide (NAD+), a CRISPR-Cas12a biosensing platform activated by DNA ligation reaction was constructed to detect ATP and NAD+. In the presence of ATP or NAD+, DNA ligase can catalyze the ligation of two oligonucleotides fragment (DNA1 and DNA2) that complement with a template, and the ligation product can activate Cas12a and generate fluorescence signal by the cleavage of single-stranded DNA reporter. This method is able to measure ATP and NAD+ in the concentration range of 0.5-100 nM and 1-200 nM, with the detection limits of 50 pM and 10 pM, respectively. In addition, this method has been successfully applied to the activity assay of T4 polynucleotide kinase (T4 PNK), and can be used to study the inhibitory effect of small molecules on T4 PNK activity. On the basis of DNA ligation reaction, CRISPR-Cas system is applied to the ultra-sensitive detection of ATP, NAD+ and T4 PNK in this work, which expands the application scope of CRISPR-Cas system in the detection of non-nucleic-acid targets.

Keywords： Analytical Chemistry CRISPR-Cas12a small biological molecules T4 polynucleotide kinase DNA ligase