In recent years, single-stranded DNA (ssDNA) has emerged as an impactful bioengineering tool, with broad-reaching applications from gene therapy to diagnostics. However, scalable synthesis of long ssDNA (>1 kb) remains a significant challenge, limiting widespread use. Conventional approaches to synthesis via solid-phase chemistry are inefficient because of purification difficulties. Promisingly, alternative enzyme-based synthesis strategies have been shown to achieve a total yield of 2 µg ssDNA from a 10 µg dsDNA template at the bench-top scale. Despite this progress, the synthesis of large quantities (>1 gram) of ssDNA using enzymatic techniques has yet to be investigated. Specifically, phospho-PCR, a highly effective enzymatic approach, uses exonuclease degradation to degrade phosphorylated DNA duplexes, generating ssDNA at relatively high yield. Here, I work towards a scalable approach via (1) small-scale optimization of phospho-PCR to reduce production cost, enabling scale-up and (2) increase reaction volume 10x. The first approach tests different cost reduction strategies focusing on minimized enzyme loading and the use of in-house reagents. Here, I found that I could attain similar ssDNA yield compared to the previously published phospho-PCR method, reducing enzyme volume, lowering reagent costs, and improving scale-up feasibility. The second approach aims to develop a 10x scale-up procedure for phospho-PCR. Specifically, I will explore effects of heat transfer on scale-up, mitigating potential negative effects via optimization of heating protocols and quenching agents. In total, these efforts have the potential to create a fast and reliable method for scaled-up long ssDNA synthesis, enabling wide-spread use of long ssDNA.
