Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 30309-0215, USA.
Anal Chem. 2010 Feb 1;82(3):1082-9. doi: 10.1021/ac902456n.
Fluorescent RNA is an important analytical tool in medical diagnostics, RNA cytochemistry, and RNA aptamer development. We have synthesized the fluorescent ribonucleotide analogue 1,3-diaza-2-oxophenothiazine-ribose-5'-triphosphate (tCTP) and tested it as substrate for T7 RNA polymerase in transcription reactions, a convenient route for generating RNA in vitro. When transcribing a guanine, T7 RNA polymerase incorporates tCTP with 2-fold higher catalytic efficiency than CTP and efficiently polymerizes additional NTPs onto the tC. Remarkably, T7 RNA polymerase does not incorporate tCTP with the same ambivalence opposite guanine and adenine with which DNA polymerases incorporate the analogous dtCTP. While several DNA polymerases discriminated against a d(tC-A) base pair only by factors <10, T7 RNA polymerase discriminates against tC-A base pair formation by factors of 40 and 300 when operating in the elongation and initiation mode, respectively. These catalytic properties make T7 RNA polymerase an ideal tool for synthesizing large fluorescent RNA, as we demonstrated by generating a approximately 800 nucleotide RNA in which every cytosine was replaced with tC.
荧光 RNA 是医学诊断、RNA 细胞化学和 RNA 适体开发的重要分析工具。我们合成了荧光核糖核苷酸类似物 1,3-二氮杂-2-氧代苯并噻嗪-核糖-5'-三磷酸（tCTP），并将其作为 T7 RNA 聚合酶在转录反应中的底物进行了测试，这是体外生成 RNA 的一种便捷途径。当转录鸟嘌呤时，T7 RNA 聚合酶以比 CTP 高 2 倍的催化效率掺入 tCTP，并有效地将额外的 NTP 聚合到 tC 上。值得注意的是，T7 RNA 聚合酶不会像 DNA 聚合酶掺入类似的 dtCTP 那样对鸟嘌呤和腺嘌呤产生相同的模棱两可的掺入。虽然一些 DNA 聚合酶对 d(tC-A)碱基对的识别仅相差<10 倍，但 T7 RNA 聚合酶在延伸和起始模式下分别对 tC-A 碱基对形成的识别相差 40 倍和 300 倍。这些催化特性使 T7 RNA 聚合酶成为合成大荧光 RNA 的理想工具，正如我们通过生成一个大约 800 个核苷酸的 RNA 来证明的那样，其中每个胞嘧啶都被 tC 取代。
