Reprogramming DNA polymerases to synthesize xeno-nucleic acids (XNAs) is an important challenge that tests current enzyme engineering tools. Here we describe an evolutionary campaign aimed at generating an XNA polymerase that can efficiently make α-l-threofuranosyl nucleic acid (TNA)—an artificial genetic polymer that is recalcitrant to nucleases and resistant to acid-mediated degradation. Starting from a homologous recombination library, iterative cycles of selection were performed to traverse the fitness landscape in search of neutral mutations with increased evolutionary potential. Subsequent directed evolution of focused mutagenic libraries yielded 10–92, a newly engineered TNA polymerase that functions with a catalytic rate of ∼1 nt s⁻¹ and >99% fidelity. A crystal structure of the closed ternary complex reveals the degree of structural change required to remodel the active site pocket for improved TNA synthesis activity. Together, these data demonstrate the importance of recombination as a strategy for evolving XNA polymerases with considerable practical value for biotechnology and medicine.

重编程 DNA 聚合酶以合成异种核酸 (XNA) 是测试当前酶工程工具的一个重要挑战。在这里，我们描述了旨在产生XNA聚合酶的进化运动，该聚合酶可以有效地制造α -l-苏呋喃糖基核酸（TNA）——一种对核酸酶不耐受并且对酸介导的降解具有抵抗力的人工遗传聚合物。从同源重组文库开始，进行迭代选择循环以遍历适应度景观，寻找具有增加的进化潜力的中性突变。随后对集中诱变文库进行定向进化，产生了 10-92，这是一种新设计的 TNA 聚合酶，其催化速率为∼ 1 nt s ^-1 ，保真度为 >99%。封闭三元复合物的晶体结构揭示了重塑活性位点袋以提高 TNA 合成活性所需的结构变化程度。总之，这些数据证明了重组作为进化 XNA 聚合酶策略的重要性，对生物技术和医学具有相当大的实用价值。