Abstract
Single-stranded DNA (ssDNA) increases the likelihood of homology directed repair with reduced cellular toxicity. However, ssDNA synthesis strategies are limited by the maximum length attainable, ranging from a few hundred nucleotides for chemical synthesis to a few thousand nucleotides for enzymatic synthesis, as well as limited control over nucleotide composition. Here, we apply purely enzymatic synthesis to generate ssDNA greater than 15 kilobases (kb) using asymmetric PCR, and illustrate the incorporation of diverse modified nucleotides for therapeutic and theranostic applications.
Highlights
Efficient Single-stranded DNA (ssDNA) synthesis on the 10+ kb-scale is a major need for numerous biotechnology applications including templated homology directed repair for genome editing[1,2,3,4], systems-scale gene synthesis and cloning[5,6,7,8,9], and scaffolded DNA origami[10,11]
Asymmetric polymerase chain reaction offers the direct synthesis of ssDNA from an underlying double-stranded DNA (dsDNA) template, unlinked to biological replication sequences, and has been applied to generate ssDNA ranging from several hundred to several thousand nucleotides in length20–23. asymmetric polymerase chain reaction (aPCR) differs from traditional PCR by having one primer in molar excess over the second primer
A general protocol was developed for the highest product yield, specific to AccuStart HiFi polymerase, showing 2 mM MgSO4 concentration, 1:50 to 1:65 reverse:forward primer ratio, 0.6 ng/μL template concentration, and up to 40 cycles (Figs S4 and S5; External Table S1), with gel extraction being used for subsequent purification (Fig. S6) yielding an average synthesis in the 1 kb range of 695 ± 35 ng (2 pmoles for a 1,000 nt ssDNA fragment) of long ssDNA per 50 μL of aPCR reaction (Fig. S7 and External Table S2)
Summary
® High-fidelity polymerases such as Phusion allow for long dsDNA synthesis in standard PCR; Phusion polymerase was unable to synthesize fragment larger than 1 kb ssDNA (Fig. 1a, lane 7 and S1, lane 7), likely due to enhanced exonuclease activity as observed with commercial genetically engineered Deep Vent (exo-) polymerase without 3′ → 5′ proofreading exonuclease activity (Fig. S2). A general protocol was developed for the highest product yield, specific to AccuStart HiFi polymerase, showing 2 mM MgSO4 concentration, 1:50 to 1:65 reverse:forward primer ratio, 0.6 ng/μL template concentration, and up to 40 cycles (Figs S4 and S5; External Table S1), with gel extraction being used for subsequent purification (Fig. S6) yielding an average synthesis in the 1 kb range of 695 ± 35 ng (2 pmoles for a 1,000 nt ssDNA fragment) of long ssDNA per 50 μL of aPCR reaction (Fig. S7 and External Table S2). The LongAmp and the LA Taq enzymes were capable of producing ssDNA products 10, 12, and 15 kb in length (Figs 1c and S10, S12–S14) While both of the enzymes were capable of synthesis of long fragments, the NEB LongAmp gave the highest yield according to gel band intensity and was reproducibly purified (50 fmoles, 20 fmoles, and 90 fmoles per 50 μL of reaction for the 10, 12, and the 15 kb, respectively) (Figs S12–S14, External Table S2). The capabilities introduced here will enable future biotechnological applications, including insertion of large chemically- or fluorescently-modified gene constructs through single gene editing experiments, long ssDNA-based digital memory storage, and scaffold-modified structured nanoparticle synthesis, amongst others
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