Codon optimization depletes stop codons in alternative reading frames of protein-coding nucleic acid therapeutics

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• Out-of-frame translation is an inherent biological process.
• Stop codon distribution in alternative reading frames dictates the length of unintended translation products.
• Codon optimization, used to enhance protein expression, often removes essential stop codons.

Purpose of the Study:

• To investigate the role of stop codons in controlling out-of-frame translation.
• To analyze stop codon distribution in natural and engineered coding sequences.
• To develop a strategy for eliminating unintended translation products in nucleic acid therapeutics.

Main Methods:

• Quantitative dual-fluorescence reporters to monitor out-of-frame translation.
• Genome-wide analysis across 10 organisms.
• Mass spectrometry to detect out-of-frame products in therapeutic sequences.

Main Results:

• Natural coding sequences have dense stop codon distributions, limiting out-of-frame products.
• Codon-optimized sequences, including mRNA vaccines, show depleted stop codons, leading to longer unintended products.
• Restoring stop codons via synonymous substitutions eliminated detectable out-of-frame products while maintaining protein integrity.

Conclusions:

• Stop codon distribution is a critical, overlooked parameter in designing synthetic nucleic acids.
• Informed sequence design can eliminate unintended translation products without altering manufacturing or regulatory processes.
• This strategy enhances the safety profile of protein-coding nucleic acid therapeutics.