Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami.

DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli. © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

Citation

Bo-Young Lee, Jaewon Lee, Dong June Ahn, Seungwoo Lee, Min-Kyu Oh. Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami. Nucleic acids research. 2021 Jun 21;49(11):6596-6603

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PMID: 34110422

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