Measuring XNA polymerase fidelity in a hydrogel particle format.

Growth in the development of engineered polymerases for synthetic biology has led to renewed interest in assays that can measure the fidelity of polymerases that are capable of synthesizing artificial genetic polymers (XNAs). Conventional approaches require purifying the XNA intermediate of a replication cycle (DNA → XNA → DNA) by denaturing polyacrylamide gel electrophoresis, which is a slow, costly, and inefficient process that requires a large-scale transcription reaction and careful extraction of the XNA strand from the gel slice. In an effort to streamline the assay, we developed a purification-free approach in which the XNA transcription and reverse transcription steps occur inside the matrix of a hydrogel-coated magnetic particle. Accordingly, a DNA primer cross-linked throughout the gel matrix is annealed to a template of defined sequence and extended with XNA. Following removal of the DNA template, the XNA product strand is copied back into DNA, recovered, amplified, cloned, and sequenced. Performing the replication cycle in the hydrogel format drastically reduces the time and reaction scales required to measure the fidelity of an XNA polymerase, making it easier to evaluate the properties of a range of candidate XNA polymerases.
(© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)