Department of Chemistry , The Scripps Research Institute , 10550 North Torrey Pines Road , La Jolla , California 92037 , United States.
School of Chemistry and Chemical Engineering , Henan Normal University , Henan 453007 , P. R. China.
J Am Chem Soc. 2019 Jul 10;141(27):10644-10653. doi: 10.1021/jacs.9b02075. Epub 2019 Jun 26.
Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.
此前，我们曾报道过一种半合成生物体（SSO）的创建，该生物体通过稳定地在其 DNA 中维持非天然碱基对（UBP），将相应的非天然核苷酸转录为 mRNA 和 tRNA 的密码子和反密码子，然后利用它们来生产含有非标准氨基酸（ncAAs）的蛋白质，从而实现信息的存储和检索。在这里，我们通过探索各种脱氧核苷酸和核糖核苷酸类似物，对 SSO 的优化工作进行了系统的扩展。重要的是，这包括首次对非天然核苷三磷酸进行体内结构-活性关系（SAR）分析。我们观察到 DNA 和 RNA 聚合酶识别非天然核苷酸的相似性和差异性，值得注意的是，我们发现各种非天然核糖核苷酸都可以有效地转录成 RNA，然后在核糖体上有效地、选择性地配对，从而介导含有 ncAAs 的蛋白质的合成。这些结果扩展了之前的研究，表明在复制过程中可以有效地、选择性地配对与天然核苷酸没有显著结构或功能同源性的核苷酸，包括信息存储和检索整个过程的每个步骤。从实际的角度来看，该结果确定了最适合复制和转录的 UBP，以及最适合转录和翻译的非天然核糖核苷三磷酸。优化后的 SSO 现在能够首次有效地生产含有多个邻近 ncAAs 的蛋白质。
