Abstract
Scalable production of kilobase single-stranded DNA (ssDNA) with sequence control has applications in therapeutics, gene synthesis and sequencing, scaffolded DNA origami, and archival DNA memory storage. Biological production of circular ssDNA (cssDNA) using M13 addresses these needs at low cost. However, one unmet goal is to minimize the essential protein coding regions of the exported DNA while maintaining its infectivity and production purity to produce sequences less than 3,000 nt in length, relevant to therapeutic and materials science applications. Toward this end, synthetic miniphage with inserts of custom sequence and size offers scalable, low-cost synthesis of cssDNA at milligram and higher scales. Here, we optimize growth conditions using an E. coli helper strain combined with a miniphage genome carrying only an f1 origin and a β-lactamase-encoding (bla) antibiotic resistance gene, enabling isolation of pure cssDNA with a minimum sequence genomic length of 1,676 nt, without requiring additional purification from contaminating DNA. Low-cost scalability of isogenic, custom-length cssDNA is demonstrated for a sequence of 2,520 nt using a bioreactor, purified with low endotoxin levels (<5 E.U./ml). We apply these exonuclease-resistant cssDNAs to the self-assembly of wireframe DNA origami objects and to encode digital information on the miniphage genome for biological amplification.
Highlights
Kilobase-length, single-stranded DNA is essential to numerous biotechnological applications including sequencing[1], cloning[2], homology directed repair templating for gene editing[3], DNA-based digital information storage[4,5], and scaffolded DNA origami[6,7,8,9]
To achieve custom sequence design of mini-scaffold DNA, helper plasmid systems are employed where the M13 coding sequences are sub-cloned onto a double-stranded, low-copy number vector that is co-transformed with a phagemid containing an single-stranded DNA (ssDNA) origin of replication (e.g., f1 origin) that allows for the synthesis and packaging of ssDNA
We show that isogenic miniphage production of circular ssDNA (cssDNA) is scalable by fermentation using the E. coli str
Summary
Kilobase-length, single-stranded DNA (ssDNA) is essential to numerous biotechnological applications including sequencing[1], cloning[2], homology directed repair templating for gene editing[3], DNA-based digital information storage[4,5], and scaffolded DNA origami[6,7,8,9]. Www.nature.com/scientificreports require subsequent purification steps for any further scale-up or would introduce background sequence contamination of the helper plasmid and dsDNA and lower yields in applications to DNA origami folding To overcome these limitations, the ssDNA origin of replication and packaging signal was entirely removed from a helper plasmid M13cp)[35], thereby enabling the biological production of isogenic cssDNA without DNA impurities Leveraging this advance, one innovative approach to achieving custom bacterial scaffolds was recently implemented using a modified M13 origin of replication, but scalability was not reported and a lack of a selection marker in the produced material indicated that scalability might be challenging[25]. We demonstrate for the first time bioreactor-scalability of production of highly pure cssDNA of less than 3,000 nt in length, which is essential for the generation of circular scaffolds for wireframe DNA origami nanoparticles with partial sequence control[19,22,23], with additional applications to write-once, read-many archival DNA data storage
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