B-255 Engineered Reverse Transcriptases and Taq DNA Polymerases Deliver Robust Yield in the Presence of Clinically Relevant Inhibitors
Authors: Walker J , Venter P , Faurholm B , Wadsworth R , Pavlica J , Marshall C , Ross C 
Published: September 2023

Abstract

Objective and Background
All biological information is read, written and edited by enzymes. If you improve the enzymes, you improve data quality for precision diagnostics. Reverse transcriptase (RT) and Taq DNA Polymerase (Taq DNAP) are commonly used to detect RNA and/or DNA viruses in patient samples for point of care (POC) diagnostics. However, crude patient samples bring over inhibitors that inhibit these enzymes leading to a decrease in enzymatic activity and an increase in false-negative results in POC testing. In addition, there is a need for faster turn-around time for patient diagnosis which is enabled by engineering more active and sensitive Taq variants that work in fast PCR set-ups. At Watchmaker Genomics, our vision is to overcome hurdles in clinical medicine by engineering enzymes that confer desired phenotypes (i.e., greater activity, thermostability, and inhibitor tolerance) that will enable more sensitive and rapid detection of diseases in clinical medicine.

Methods
We take a 3-pronged approach to protein engineering: rational design, computational design and directed evolution. The aforementioned engineering approaches were used to rapidly identify and characterize &gt; 20 RT and Taq DNAP variants. First, we deeply characterized each RT variant by measuring processivity via cDNA length and by measuring inhibitor tolerance and thermostability via RT-qPCR. In RT-qPCR, first strand synthesis (FSS) was done at temperatures from 42–65°C in the presence and absence of various clinically relevant inhibitors. For measuring inhibitor tolerance the ΔCt was calculated to determine the level of inhibition (ΔCt = Ct (with inhibitor) - Ct (without inhibitor)). Next, we characterized the inhibitor tolerance of the Taq variants by performing qPCR reactions in the presence of clinically relevant inhibitors. To determine if the Taq variant would be an appropriate fit for fast PCR, the polymerization activity of the Taq DNAP variants were characterized by annealing a single primer to ssDNA and allowing isothermal extension of the primer while monitoring the SYBR green fluorescence increase.

Results and Conclusions
Reverse transcriptases termed; StellarScript, StellarScript HT and StellarScript HT+ exhibit high processivity at 42°C. StellarScript HT+ exhibited the highest inhibitor tolerance, processivity and thermostability at temperatures up to 65°C. Based on these key attributes, StellarScript HT+ is well suited to give robust yields with clinical samples. Our Taq DNAP variants exhibited a 2.26 fold increase in polymerase activity over native Taq DNAP. Taq variants were identified that efficiently and robustly (i.e., high yield) amplify DNA in the presence of inhibitors when compared to native Taq DNAP. Based on the increased polymerase activity and inhibitor tolerance, WMG Taq variants are well suited to give robust DNA yields with clinical samples. We were successful in engineering RT and Taq DNAP variants that are highly thermostable, active and inhibitor tolerant and thus will enable breakthroughs in existing POC diagnostics.