Design and off-target prediction for antisense oligomers targeting bacterial mRNAs with the MASON webserver

Abstract

ABSTRACTAntisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (MakeAntiSenseOligomersNow), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON’s off-target predictions on experiments in which we treatedSalmonella entericaserovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential geneacpPbut carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to targetacpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenicEscherichia coli(UPEC) treated with eleven different PNAs confirmed our findings are not unique toSalmonella. We believe that MASON’s off-target assessment will improve the design of specific PNAs and other ASOs.