Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States.
Synthorx, a Sanofi Company, La Jolla, California 92037, United States.
J Am Chem Soc. 2020 Nov 11;142(45):19029-19032. doi: 10.1021/jacs.0c09230. Epub 2020 Oct 29.
Through the development of unnatural base pairs that are compatible with native DNA and RNA polymerases and the ribosome, we have expanded the genetic alphabet and enabled in vitro and in vivo production of proteins containing noncanonical amino acids. However, the absence of assays to characterize transcription has prevented the deconvolution of the contributions of transcription and translation to the reduced performance of some unnatural codons. Here we show that RNA containing the unnatural nucleotides is efficiently reverse transcribed into cDNA, and we develop an assay to measure the combined fidelity of transcription and reverse transcription. With this assay, we examine the performance of a wide variety of unnatural codons, both in vitro and in the in vivo environment of a semisynthetic organism. We find that transcription is generally efficient, decoding at the ribosome is generally more challenging, and, correspondingly, sequence-dependent translation efficiency is the origin of variable codon performance.
通过开发与天然 DNA 和 RNA 聚合酶以及核糖体兼容的非天然碱基对，我们扩展了遗传密码子，并能够在体外和体内生产含有非规范氨基酸的蛋白质。然而，缺乏用于表征转录的测定方法，使得无法剖析转录和翻译对某些非天然密码子性能降低的贡献。在这里，我们表明含有非天然核苷酸的 RNA 可以有效地逆转录成 cDNA，并且我们开发了一种测定方法来测量转录和逆转录的综合保真度。使用该测定法，我们在半合成生物体的体内和体外环境中检查了各种非天然密码子的性能。我们发现转录通常是高效的，核糖体上的解码通常更具挑战性，相应地，序列依赖性翻译效率是可变密码子性能的起源。
Zotero 插件