(1055-B) In vitro evolution of T7 RNA polymerase facilitated by droplet microfluidics

Abstract: Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. The flagship platform to replace the phosphoramidite synthesis process is envisioned to be template-independent catalysis by terminal deoxynucleotidyl transferase, although specific conditions are needed to achieve controllable DNA synthesis. Among template-directed methods, asymmetric PCR (aPCR) is suggested as a means to synthesize long ssDNA products, although several reports indicate that the initial optimization of aPCR is not easy and different ssDNAs cannot be produced using a single protocol. Here, we suggest considering in vitro transcription (IVT) using highly processive T7 RNA polymerase (T7 RNAP) as an alternative approach for isothermal synthesis of ssDNA.
To achieve that, we engineered T7 RNA polymerase to incorporate deoxynucleotide triphosphates (dNTPs) so that ssDNA is produced directly by IVT. We performed in vitro evolution employing droplet microfluidics. Briefly, E. coli cells expressing mutant T7 RNAP variants were encapsulated in droplets together with lysis and IVT reagents, as well as dNTPs and a fluorescent reporter. After the in-droplet IVT assay, fluorescence-activated droplet sorting was used to enrich the variants able to produce transcripts utilizing dNTPs. We identified mutations V783M, V783L, V689Q, and G555L as novel T7 RNAP variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates.