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

Area of Science:

• Microbiology
• Molecular Biology
• Bioinformatics

Background:

• Antisense oligomers (ASOs), including peptide nucleic acids (PNAs), are being developed as sequence-specific RNA antibiotics to inhibit bacterial gene translation.
• A significant challenge in ASO development is minimizing off-target binding to unintended transcripts, which can lead to unwanted side effects.

Purpose of the Study:

• To develop a computational tool, MASON (make antisense oligomers now), to aid in the design of PNAs with reduced off-target effects.
• To improve the specificity of PNAs targeting bacterial messenger RNAs (mRNAs).

Main Methods:

• Development of the MASON web server for designing PNAs complementary to bacterial mRNA translational start sites.
• Experimental validation using *Salmonella enterica* serovar Typhimurium treated with modified PNAs targeting the essential *acpP* gene.
• RNA-sequencing (RNA-seq) analysis to assess gene expression changes and identify off-target binding.

Main Results:

• MASON facilitates the design of PNAs and predicts potential off-target sites.
• Experiments revealed that PNAs with terminal mismatches targeting *acpP* still exhibited binding, indicating broader off-target effects than initially expected.
• Findings were corroborated using RNA-seq data from *Escherichia coli* (UPEC), demonstrating the generalizability of the off-target phenomenon.

Conclusions:

• MASON's off-target assessment capabilities can enhance the design of specific PNAs and other antisense oligomers.
• Understanding and mitigating off-target effects are crucial for the successful development of PNA-based therapeutics.