Download or read book Development of Riboswitch-based Sensors for High-throughput Enzyme Activity Screens written by Yichi Su and published by . This book was released on 2018 with total page 161 pages. Available in PDF, EPUB and Kindle. Book excerpt: Development of robust assay reagents for high-throughput enzyme activity screens is essential for biomedical research and drug development. However, conventional protein-based assay reagents have their limitations, including difficulty in engineering, limited signal dynamic range, lack of small molecule specificity and batch-to-batch variation. Most importantly, few assay reagents are applicable to both in vitro and live cell settings. An attractive alternative that overcomes these limitations is riboswitch-based sensors. Riboswitches are cis-regulatory structured RNA elements capable of controlling expression of downstream genes by binding to small molecule ligands. The naturally evolved RNA elements retain the advantages of synthetic aptamers, including ease of engineering, low cost in synthesis, and high batch-to-batch reproducibility, while they incorporate additional desirable features including remarkable affinity and selectivity for small molecule ligands, high-fidelity folding and thermostability for functioning in cellular environment. One strategy to engineer riboswitch-based sensor is to fuse the canonical terminal stem of the riboswitch aptamer domain to an internal stem of a signaling aptamer. Using this strategy, several prototypical riboswitch-based sensors were developed to detect essential metabolites and second messengers both in vitro and in live bacteria. However, despite their great potential, the universality of riboswitch-based sensors is limited by the number and the diverse topologies of natural riboswitch classes. Herein, novel strategies for engineering riboswitch-based sensors for more diverse ligands with improved sensitivity and specificity are described. First, in order to engineer sensors based on pseudoknot-containing riboswitches, to which the conventional engineering strategy was not applicable, an alternative inverted fusion strategy was invented. Riboswitch-based fluorescent biosensors for an essential metabolite S-adenosyl-L-homocysteine(SAH) were developed using this strategy. These SAH biosensors provide a direct “mix and go” high-throughput activity assay for methyltransferases, and a way to monitor the increase of SAH levels in live Escherichia coli upon chemical inhibition of methylthioadenosine nucleosidase (MTAN), an enzyme that is involved in quorum-sensing. Then, in order to engineer riboswitch-based sensors to interrogate ligands that are structural analogues to natural riboswitch ligands, a pipeline involving phylogenetic screening and structure-guided rational design was demonstrated on engineering bacterial 3'3'-cyclic di-GMP (cdiG) riboswitch-based biosensors into sensing a mammalian immune pathway second messenger 2'3'-cyclic-AMP-GMP (cGAMP). The resulting biosensors enabled in vitro high-throughput activity screening assays for cGAMP synthase, and direct cGAMP quantitation in dsDNA-stimulated L929 cell lysates. Finally, aiming to further expand the target ligand scope of riboswitch-based sensors to virtually any given small molecule ligands, two novel mechanism-based in vitro selection strategies, termed Capture-SELEX and Dye-SELEX, were explored. Proof-of-concept experiments showcased their capability of enriching sequences with desirable sensing properties from random RNA counterparts. It is envisioned that the strategies described here will forward the development of new riboswitch-based sensors as competitive assay reagents to antibodies, and eventually advancing basic biological understanding and biomedical applications.