Unintended Creation or Insertion of Antisense Promoter Motifs During Codon Optimization: A Cyber-Biosecurity Risk

Area of Science:

• Synthetic Biology
• Molecular Biology
• Bioinformatics

Background:

• Codon optimization enhances protein expression via synonymous codon substitutions.
• Traditional methods overlook impacts on the complementary DNA strand.
• Antisense motifs, like bacterial promoters, can be inadvertently introduced.

Purpose of the Study:

• Investigate inadvertent introduction of antisense promoter motifs during codon optimization.
• Assess the feasibility of silent insertion of these motifs without altering protein output.
• Develop computational tools for scanning and defense against these vulnerabilities.

Main Methods:

• Developed a computational pipeline for scanning DNA sequences for antisense motifs.
• Implemented a silent insertion algorithm preserving amino acid sequences.
• Evaluated insertion feasibility across a large genomic dataset (484,741 sequences).

Main Results:

• 77.28% of motif-free sequences allowed silent insertion of antisense motifs.
• Only 4.8% of sequences naturally contained such motifs.
• Identified a novel cyber-biosecurity vulnerability in DNA design pipelines.

Conclusions:

• Codon optimization requires bi-directional screening for both forward and antisense strands.
• Silent insertion of antisense motifs presents a potential risk in synthetic DNA.
• Developed open-source software to mitigate these risks in academic and industrial settings.