Evaluating the Rate and Substrate Specificity of Laboratory Evolved XNA Polymerases

Engineered polymerases that can copy genetic information between DNA and xeno-nucleic acids (XNA) hold tremendous value as reagents in future biotechnology applications. However, current XNA polymerases function with inferior activity relative to their natural counterparts, indicating that current polymerase engineering efforts would benefit from new benchmarking assays. Here, we describe a highly parallel, low-cost method for measuring the average rate and substrate specificity of XNA polymerases in a standard qPCR instrument. Our approach, termed polymerase kinetic profiling (PKPro), involves monitoring XNA synthesis on a self-priming template using high-resolution melting (HRM) fluorescent dyes that intercalate into the growing duplex as the template strand is copied into XNA. Since changes in fluorescence are directly proportional to XNA synthesis, quantitative measurements are obtained by calibrating the fluorescent signal against chemically synthesized standards. Using PKPro, we discovered that XNA polymerases function with rates of ∼1-80 nt/min and exhibit substrate specificities of ∼0.1-5-fold for xNTP versus dNTP. Last, we show how PKPro could be used in a highly parallel screen by analyzing 288 different polymerase reaction conditions. On the basis of these results, we suggest that PKPro provides a powerful tool for evaluating the activity of XNA polymerases.