Characterization of a novel PrimPol from Thermus thermophilus

Trabajo presentado en el 6th DNA Polymerases meeting, celebrado en Stockholm (Suecia) del 22 al 25 de mayo de 2022.Accurate DNA amplification has become essential for most molecular diagnostic and therapeutic techniques. With the recent approvals of gene therapy products, this fast-growing field is facing an increased demand for large-scale production and manufacturing of clinical grade synthetic DNA. TruePrime¿, commercialized by 4BaseBio S.L. biotech, is an isothermal DNA amplification method based on the combination of two DNA-synthesizing enzymes. A thermostable DNA primase called Thermus thermophilus PrimPol (TthPrimPol)1 synthesizes DNA primers, unlike conventional primases, which only make RNA primers. These DNA primers are then elongated by the highly processive and strand-displacement profficient bacteriophage Phi29 DNA polymerase (Phi29 DNApol), which allows an isothermal and exponential cascade of multiple displacement amplification (MDA). Both enzymes can work in the presence of Mg2+ as cofactor, a requisite to achieve faithful DNA synthesis by Phi29 DNApol. The combination of both enzymes in TruePrime¿ has demonstrated advantages such as great genomic coverage, minute DNA sensitivity, high amplification product, low chimera formation, or high single nucleotide variants (SNV) detection1. Nevertheless, different approaches are being pursued in attempts to optimize this technology. Here* we describe the in silico identification, using amino acid similarity search, of a new putative PrimPol that we termed TthPrimPol2, its purification, and its preliminary in vitro characterization. This new enzyme is a bona fide PrimPol as it preferentially synthesizes DNA primers. Its high efficiency when priming results from a higher binding affinity for ssDNA templates. Accordingly, it generates longer primers of an average size of 13-15 dNTPs long. As a thermostable enzyme, its primase activity is stronger when increasing the temperature up to 50 OC. Importantly, Phi29 DNApol can processively elongate the DNA primers generated by TthPrimPol2 when combined. Future studies will confirm whether its use as a component of TruePrime¿ can improve genome coverage evenness, DNA sensitivity, or yield efficiency, still maintaining exceptionally low error rates.Industrial PhD funding IND2018/IND-998