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Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR).

ABSTRACT: In vitro selection of ligand-responsive ribozymes can identify rare, functional sequences from large libraries. While powerful, key caveats of this approach include lengthy and demanding experimental workflows; unpredictable experimental outcomes and unknown functionality of enriched sequences in vivo. To address the first of these limitations, we developed Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR). LigASERR is scalable, amenable to automation and requires less time to implement compared to alternative methods. To improve the predictability of experiments, we modeled the underlying selection process, predicting experimental outcomes based on sequence and population parameters. We applied this new methodology and model to the enrichment of a known, in vitro-selected sequence from a bespoke library. Prior to implementing selection, conditions were optimized and target sequence dynamics accurately predicted for the majority of the experiment. In addition to enriching the target sequence, we identified two new, theophylline-activated ribozymes. Notably, all three sequences yielded riboswitches functional in Escherichia coli, suggesting LigASERR and similar in vitro selection methods can be utilized for generating functional riboswitches in this organism.

SUBMITTER: Haines MC

PROVIDER: S-EPMC7445825 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:S-Glutathionylation (SSG) is an important regulatory posttranslational modification on protein cysteine (Cys) thiols, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols, and covalent capture of reduced thiols using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was initially validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG modification compared to controls. This approach was extended to identify potential SSG-modified Cys sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment, thus providing a database of proteins and Cys sites susceptible to this modification under oxidative stress. Functional analysis revealed that the most significantly enriched molecular function categories for proteins sensitive to SSG modifications were free radical scavenging and cell death/survival. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of SSG-modified proteins. The analytical strategy also provides a unique approach to determining the major pathways and cellular processes most susceptible to S-glutathionylation under stress conditions.

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Riboswitch identification using Ligase-Assisted Selection for the Enrichment of Responsive Ribozymes (LigASERR).

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