Robinson, N. E. | bioRxiv Science

bioRxiv2026

Rapid in vitro synthesis of DNA templates via Sidewinder for polyadenylated Hantavirus mRNA vaccine candidates

As the recent COVID-19 pandemic illustrated, zoonotic viruses and other pathogens pose a credible threat to public health. Recent advancements in vaccine technology, particularly mRNA vaccines, provide key tools for an effective and swift public health response. Although mRNA vaccines can be developed more quickly than traditional vaccines, fast and accurate construction of DNA templates for these vaccines remains a critical bottleneck. Using our novel DNA assembly technology, Sidewinder, we rapidly designed and built multiple mRNA vaccine candidates to guard against a potential outbreak of Hantavirus (ANDV). We successfully constructed the DNA templates from oligo pools and produced the mRNA for three vaccine candidates in just 2 days after delivery of the synthetic DNA oligos.

biochemistry↗

bioRxiv2026

One-pot parallel Sidewinder construction from oligo pools

Reliable and cost-effective de novo DNA production at scale has become increasingly important to studying and engineering biology. Double-stranded DNA is constructed from short synthetic single-stranded DNA oligonucleotides synthesized either individually or as a pool. Oligonucleotide pools offer substantially reduced costs at the sacrifice of yield and individual oligo isolation, making efficient multiplexed DNA construction from pools a complex engineering challenge, yet one which promises to reduce cost, labor, and turnaround time if solved. Here we introduce Oligo Pool Sidewinder assembly, which enables one-pot, parallel assembly of hundreds of DNA fragments simultaneously into dozens of defined constructs with high fidelity from oligo pools. We pair a computational workflow for string-based bespoke oligo design with a set of construction rules for highly multiplexed assembly. We demonstrate recovery of individual sequences from the pool by construct-specific amplification with misconnection rates as low as 1 in 10. This high fidelity for connection enables hundreds of oligos to co-assemble in a single reaction. Further, we show universal amplification of libraries of defined target sequences in a single PCR and extend the approach through in vitro hierarchical assembly to construct a large 12.5-kilobase synthetic linear DNA product.

molecular biology↗