A fluorescence-based high-throughput screening method for determining the activity of diguanylate cyclases and c-di-GMP phosphodiesterases. Issue 33 (21st May 2020)

• Record Type: Journal Article
• Authors: Wang, Zi-Qiang; Xuan, Teng-Fei; Liu, Jun; Chen, Wei-Min; Lin, Jing
• Is Part Of: RSC advances. Volume 10:Issue 33(2020)
• Page Start: 19482
• Page End: 19489
• Publication Date: 2020-05-21
• Subjects: Chemistry -- Periodicals; 540.5
• DOI: 10.1039/d0ra02540b
• Languages: English
• ISSNs: 2046-2069

Abstract

Abstract : The dinucleotide 3′, 5′-cyclic diguanylic acid (c-di-GMP) is a critical second messenger found in bacteria. Abstract : The dinucleotide 3′, 5′-cyclic diguanylic acid (c-di-GMP) is a critical second messenger found in bacteria. High cellular levels of c-di-GMP are associated with a sessile, biofilm lifestyle in many bacteria, which is associated with more than 70% of clinically resistant infections. Cellular c-di-GMP concentrations are regulated by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which are responsible for the production and degradation, respectively, of c-di-GMP. Therefore, DGCs and PDEs might be attractive drug targets for controlling biofilm formation. In this study, a simple and universal high-throughput method based on a c-di-GMP-specific fluorescent probe for the determination of DGC and PDE activity was described. By using the proposed method, the c-di-GMP content in samples was rapidly quantified by measuring the fluorescence intensity in a 96-well plate by using a microplate reader. In addition, the probe molecule A18 directly interacted with the substrate c-di-GMP, and the method was not limited by the structure of enzymes.