大规模合成长链RNA的简易低成本工艺

A Simple and Cost Effective Process for Large-scale Production of Long Oligoribonucleotides

由于现有技术所限,RNA分子长度和二级结构往往成为RNA合成困难的主要原因。提供了一种简单低成本大规模制备和纯化长链RNA药物的新工艺,尤其针对具有稳定二级结构的长链RNA药物。采用引物延伸方法替代PCR和线性质粒DNA方法制备线性DNA模板可减少步骤及降低污染,然后用T7 RNA聚合酶转录制备的甲氧基修饰的线性DNA模板获得高均一度的长链RNA,转录粗产物直接用source 15Q阴离子HPLC分离T7 RNA聚合酶、rNTP、转录中断产物、内毒素和模板DNA等,从而获得高纯度RNA终产物。该工艺无需繁琐的酚/氯仿抽提和RNA变性,尤其适用于RNA的大量制备。

The length and stable secondary structure of RNA molecule are general obstacles in RNA synthesis because of current technological bottlenecks. A simple and cost effective process for large-scale preparation and purification of long oligoribonucleotides with stable secondary structure was presented. High homogeneous RNAs are transcribed in vitro with T7 RNA polymerase using linear 2＇-Ome modified DNA templates, which were prepared by primer extension instead of PCR amplification or linearized plasmid DNA transcription to reduce contamination. The crude transcripts are then directly subjected to an anion-exchange HPLC using source 15Q to separate T7 RNA polymerase, unincorporated rNTPs, small abortive transcripts, endotoxin and DNA templates from pure RNA products. The novel process does neither require tedious phenol/ chloroform extraction nor denaturation of RNA, which is especially useful for larger RNAs preparations.