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

Codon optimization is a cornerstone technique in synthetic biology and biotechnological production, aimed at enhancing heterologous protein expression through synonymous codon substitutions. While optimization traditionally focuses on forward-strand translation efficiency, its impact on the complementary DNA strand is not always examined carefully enough. In this study, we investigate whether codon optimization inadvertently introduces antisense motifs, specifically bacterial antisense promoter (e.g., ‘TATAAT’), and whether such motifs can be silently injected into coding sequences on purpose without altering protein output. We developed a computational pipeline that (i) scans optimized sequences for antisense motifs, unintended; (ii) implements a silent injection algorithm that preserves amino acid sequence; and (iii) evaluates injection feasibility across a large genomic dataset. In a dataset of 484,741 protein-coding sequences, only 4.8 % naturally contained the motif, yet 77.28 % of motif-free sequences permitted silent injections. We extend these findings with codon bias analysis, derive analytical bounds for injection complexity, and propose computational defenses. These results uncover a novel cyber-biosecurity vulnerability in DNA design pipelines, emphasizing the need for bi-directional screening in codon optimization tools.