Expanding the Genetic Alphabet by Design and Selection

描述（由申请人提供）：DNA和RNA的信息存储潜力受到天然碱基对的选择性氢键（H-键合）的限制。我们的目标是通过设计非天然碱基对和进化识别它们的聚合酶来规避这一限制。我们一直在探索碱基对的相互作用是由分子间的力量，而不是H-键，包括疏水性，其对蛋白质结构的贡献已被赞赏了几十年。在上一个资助期间，我们成功地完成了我们的具体目标：1）合成和表征非天然碱基对; 2）建立一个选择系统，以进化接受非天然底物的DNA聚合酶。从这项工作中，已经开发了几种复制系统，包括基于两个3-氟苯核苷（3FB）之间形成的“自对”的复制系统。3FB自配对是第一个非天然碱基对，它是针对所有可能的错配对进行选择性合成的。初步的结构表征表明，自配对介导的偶极-偶极力和一个有趣的碱基间氟-氢键。我们还开发了一种基于活性的选择系统，并在三个单独的应用中显示它能够进化合成非天然DNA的DNA聚合酶。我们现在建议在这些成功的基础上，首先描述3FB自对和错对的结构和复制，并使用这些数据来帮助设计更好的自对。我们还将进化出专门用于复制含有3FB自我对的DNA的DNA聚合酶。最后，我们将开始探索将自配对转录成非天然RNA，这是扩展生物遗传密码的下一步，为半合成生物奠定基础。

DESCRIPTION (provided by applicant): The information storage potential of DNA and RNA is limited by the selective hydrogen bonding (H-bonding) of the natural base pairs. We aim to circumvent this restriction by designing unnatural base pairs and evolving polymerases that recognize them. We have been exploring base pairs whose interactions are driven by intermolecular forces other than H-bonding, including hydrophobicity, whose contribution to protein structure has been appreciated for decades. During the previous funding period, we successfully completed our specific aims to 1) synthesize and characterize unnatural base pairs; and 2) establish a selection system to evolve DNA polymerases that accept the unnatural substrates. From this work, several replication systems have been developed, including one based on 'self-pairs' formed between two 3-fluorobenzene nucleosides (3FB). The 3FB self-pair is the first unnatural base pair that is processively synthesized with selectivity against all possible mispairs. Preliminary structural characterization implies that the self-pairing is mediated by dipole-dipole forces and an interesting interbase fluorine-hydrogen bond. We have also developed an activity based selection system and in three separate applications shown it to be capable of evolving DNA polymerases that synthesize unnatural DNA. We now propose to build on these successes by first characterizing the structure and replication of 3FB self-pairs and mispairs, and using this data to aid in the design of a better self-pair. We will also evolve DNA polymerases specifically tailored to replicate DNA containing 3FB self-pairs. Finally, we will begin to explore the transcription of the self-pair into unnatural RNA, the next step toward expanding an organism's genetic code, laying the foundation for a semi-synthetic organism.