biostudies-literatureFuller JDefense Advanced Research Projects AgencyJoint Program Executive Office for Chemical, Biological, Radiological and Nuclear DefenseNIAID NIH HHSNational Cancer InstituteNCI NIH HHSNIH/NIAID Collaborative Influenza Vaccine Innovation Centers9https://www.ebi.ac.uk/biostudies/studies/S-EPMC12800232biostudies-literatureUnknown11(1)DNA vaccines have garnered considerable attention due to their recent success in humans for SARS-CoV-2 and immunotherapy for cancer. However, conventional methods for creating and manufacturing DNA vaccines at-scale are slow and rate-limiting for timely response. Herein, we introduce a rapid and completely synthetic workflow that harnesses enzymes to create bulk DNA from a sequence text file. This synthetic workflow termed Enzymatic DNA Synthesis & Rolling-Circle Amplification (EDS-RCA) leverages multiple enzymes to print DNA oligos and assemble them into genes prior to cloning into circular constructs for rolling-circle amplification (RCA). We show that the resulting EDS-RCA DNA elicits comparable vaccine immunogenicity as standard plasmid format, despite the DNA being a large concatemeric repeat. The EDS-RCA method generated the hemagglutinin gene of H1N1 at a mean per-base error rate as low as ~1 mutation every 10,000 bases and, upon DNA vaccination, elicited strong antibody and cellular immune responses. Skin delivery of EDS-DNA using gene gun facilitated striking vaccine dose-sparing capabilities in comparison to intramuscular electroporation methods. In total, DNA vaccines produced by EDS-RCA are immunogenic and amenable to numerous delivery-modalities with preclinical mouse models and could offer an alternative for rapid scale-up of DNA vaccines for future human use.NPJ vaccinesNovel enzymatic DNA produced from a text file achieves comparable immune responses as plasmid vaccine.PMC1280023275N93019C00051N66001-21-C-4014T32CA009171T32 CA009171HR0011-21-9-0001Weiner DBRyan RNelson JFuller DHGodron XKvam ECreton SGriffin WHall CTursi NJFuller JTimp WBlatney KfalseNovel enzymatic DNA produced from a text file achieves comparable immune responses as plasmid vaccine.DNA vaccines have garnered considerable attention due to their recent success in humans for SARS-CoV-2 and immunotherapy for cancer. However, conventional methods for creating and manufacturing DNA vaccines at-scale are slow and rate-limiting for timely response. Herein, we introduce a rapid and completely synthetic workflow that harnesses enzymes to create bulk DNA from a sequence text file. This synthetic workflow termed Enzymatic DNA Synthesis & Rolling-Circle Amplification (EDS-RCA) leverages multiple enzymes to print DNA oligos and assemble them into genes prior to cloning into circular constructs for rolling-circle amplification (RCA). We show that the resulting EDS-RCA DNA elicits comparable vaccine immunogenicity as standard plasmid format, despite the DNA being a large concatemeric repeat. The EDS-RCA method generated the hemagglutinin gene of H1N1 at a mean per-base error rate as low as ~1 mutation every 10,000 bases and, upon DNA vaccination, elicited strong antibody and cellular immune responses. Skin delivery of EDS-DNA using gene gun facilitated striking vaccine dose-sparing capabilities in comparison to intramuscular electroporation methods. In total, DNA vaccines produced by EDS-RCA are immunogenic and amenable to numerous delivery-modalities with preclinical mouse models and could offer an alternative for rapid scale-up of DNA vaccines for future human use.2025-01-01T00:00:00Z2025 Dec2026-06-11T04:59:16.775Z2026-06-11T03:08:27.651ZS-EPMC128002324139077410.1038/s41541-025-01329-0