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| A Simple and Cost Effective Process for Large-scale Production of Long Oligoribonucleotides |
| ZHANG Ping-jing1,2, LI Tie-jun1,2, ZHOU Song-feng1,2, ZHU Yuan-yuan1,2,3, CHEN Jian-xin1,2,3, LU Yi-xiang1,2, WEN Feng1,2 |
1. Biomics Biotechnologies Co. Ltd, Nantong 226016, China;
2. Small RNA Technology and Application Institute, Nantong University, Nantong 226016, China;
3. School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China |
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Abstract 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.
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Received: 16 January 2013
Published: 25 April 2013
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