Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China.
Shenzhen Huazhong University of Science and Technology Research Institute, Shenzhen, China.
RNA Biol. 2022 Jan;19(1):1130-1142. doi: 10.1080/15476286.2022.2139113.
RNA research and applications are underpinned by transcription (IVT), but RNA impurities resulting from the enzymatic reagents severely impede downstream applications. To improve the stability and purity of synthesized RNA, we have characterized a novel single-subunit RNA polymerase (RNAP) encoded by the psychrophilic phage VSW-3 from a plateau lake. The VSW-3 RNAP is capable of carrying out RNA synthesis at low temperatures (4-25°C). Compared to routinely used T7 RNAP, VSW-3 RNAP provides a similar yield of transcripts but is insensitive to class II transcription terminators and synthesizes RNA without redundant 3'-cis extensions. More importantly, through dot-blot detection with the J2 monoclonal antibody, we found that the RNA products synthesized by VSW-3 RNAP contained a much lower amount of double-stranded RNA byproducts (dsRNA), which are produced by transcription from both directions and are significant in T7 RNAP IVT products. Taken together, the VSW-3 RNAP almost eliminates both terminal loop-back dsRNA and full-length dsRNA in IVT and thus is especially advantageous for producing RNA for use.
RNA 的研究和应用依赖于转录（IVT），但酶试剂产生的 RNA 杂质严重阻碍了下游应用。为了提高合成 RNA 的稳定性和纯度，我们对来自高原湖泊的嗜冷噬菌体 VSW-3 编码的新型单亚基 RNA 聚合酶（RNAP）进行了表征。VSW-3 RNAP 能够在低温下（4-25°C）进行 RNA 合成。与常规使用的 T7 RNAP 相比，VSW-3 RNAP 提供了相似的转录本产量，但对 II 类转录终止子不敏感，并且合成没有冗余 3'-顺式延伸的 RNA。更重要的是，通过 J2 单克隆抗体的斑点印迹检测，我们发现 VSW-3 RNAP 合成的 RNA 产物中双链 RNA 副产物（dsRNA）的含量要低得多，dsRNA 是由双向转录产生的，在 T7 RNAP IVT 产物中非常显著。总之，VSW-3 RNAP 几乎消除了 IVT 中末端环回双链 RNA 和全长双链 RNA，因此特别有利于生产用于研究的 RNA。
