Ong, J. Y. | Search bioRxiv

bioRxiv2021

Synonymous Mutation Generator: a web tool for designing RNAi-resistant sequences

RNA interference (RNAi) is a useful technique for knocking down a protein of interest, allowing for the study of the function of a gene product. However, RNAi techniques are prone to off-target effects, such as non-specific knockdown of genes besides the protein of interest. An important control and companion to RNAi knockdown experiments is the rescue experiment, wherein gene function is restored by expression of an RNAi-resistant construct of the protein of interest. Generating an RNAi-resistant construct of the protein of interest involves generating silent mutations within the coding sequence of the protein so that the resulting amino acid product is the same, but the protein mRNA is no longer a target for the RNAi. Here, Synonymous Mutation Generator, a Python-based web tool that takes an input DNA coding sequence and outputs a synonymous DNA coding sequence that is RNAi-resistant, is described. This web tool should be a useful resource for researchers cloning RNAi-resistant constructs. Synonymous Mutation Generator is easy to use and can be found at jong2.pythonanywhere.com, and the source code is available on GitHub.

molecular biology↗

bioRxiv2020

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of weeks. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methylmethane sulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to MMS demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain but with lower sensitivity than the plate-based assay. In conclusion, we propose yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.View Full Text

pharmacology and toxicology↗