近日，美国哈佛医学院George M. Church等研究人员合作报道了RNA寡核苷酸的模板非依赖酶法合成。2024年7月12日，《自然—生物技术》杂志在线发表了这项成果。

研究人员报告了一种基于水相、模板无关的酶促RNA寡核苷酸合成平台的开发和优化，作为传统化学方法的替代。RNA寡核苷酸的酶促合成通过使用新的CID1聚(U)聚合酶突变体，控制性地引入具有常见的3′-O-烯丙基醚阻断基的可逆终止核苷酸实现。研究人员实现了平均95%的偶联效率，并展示了十个完整的液相合成周期，用以产生天然和治疗相关的修饰序列。

接着，研究人员在固相上定性评估了该平台，进行了几种N+5寡核苷酸的酶促合成，使用了控制孔玻璃支持。采用基于水相的过程将提供关键优势，包括减少溶剂使用和可持续的治疗性寡核苷酸制造。

据悉，RNA寡核苷酸已成为治疗疾病的强大治疗方式，然而目前的制造方法可能无法满足未来预期的需求。

附：英文原文

Title: Template-independent enzymatic synthesis of RNA oligonucleotides

Author: Wiegand, Daniel J., Rittichier, Jonathan, Meyer, Ella, Lee, Howon, Conway, Nicholas J., Ahlstedt, Daniel, Yurtsever, Zeynep, Rainone, Dominic, Kuru, Erkin, Church, George M.

Issue&Volume: 2024-07-12

Abstract: RNA oligonucleotides have emerged as a powerful therapeutic modality to treat disease, yet current manufacturing methods may not be able to deliver on anticipated future demand. Here, we report the development and optimization of an aqueous-based, template-independent enzymatic RNA oligonucleotide synthesis platform as an alternative to traditional chemical methods. The enzymatic synthesis of RNA oligonucleotides is made possible by controlled incorporation of reversible terminator nucleotides with a common 3′-O-allyl ether blocking group using new CID1 poly(U) polymerase mutant variants. We achieved an average coupling efficiency of 95% and demonstrated ten full cycles of liquid phase synthesis to produce natural and therapeutically relevant modified sequences. We then qualitatively assessed the platform on a solid phase, performing enzymatic synthesis of several N+5 oligonucleotides on a controlled-pore glass support. Adoption of an aqueous-based process will offer key advantages including the reduction of solvent use and sustainable therapeutic oligonucleotide manufacturing.

DOI: 10.1038/s41587-024-02244-w

Source: https://www.nature.com/articles/s41587-024-02244-w