Expanding the Genetic Alphabet by Design and Selection

所有生物的多样性都编码在它们的DNA中， 通过DNA复制稳定维持，然后通过 转录成RNA和翻译成蛋白质。因此， 生命受到组成DNA和RNA的四种天然核苷酸的限制 以及它们所编码的20种天然氨基酸。被潜在的 化学家和生物学家一直着迷于 扩展遗传字母表（DNA和RNA）以及遗传密码的想法 （蛋白质）。这需要一个非自然的碱基对，在DNA中复制，转录， 进入RNA，然后能够将非天然氨基酸掺入到 蛋白质翻译基因字母表/编码的扩展将使 DNA，RNA和蛋白质被定制为具有新的特性-就像这些 生物聚合物正受到越来越多的关注， 材料到治疗。合成或进化这些生物聚合物的能力， 超出其自然成分编码范围的期望活动承诺， 大大增加了其潜在的应用。遗传字母表/密码的扩展 也为第一个半合成生物体奠定了基础， 增加其DNA中的信息，并以新蛋白质的形式将其检索出来。一项调查 的文献表明，遗传字母表/代码的扩展，至少在 目前由于缺乏功能性非天然碱基对， 以足够的效率和保真度复制和转录。 在上一个资助期间，我们开发了第一个非自然碱基对， d5 SICS：dNaM，其以高效和保真度复制和转录， 开始接近自然碱基对的水平。分子识别在这对 不像天然碱基对那样基于互补氢键，而是基于 互补的疏水和包装力，更类似于蛋白质。我们也 开发了一种聚合酶选择系统，能够定制负责 DNA的复制以更好地识别非天然碱基对。有了这些工具， 另一方面，我们现在建议使用各种合成和生物方法：1） 表征d5 SICS的结构决定因素：dNaM稳定性、复制和 2）优化d5 SICS：dNaM用于天然样复制和转录; 和3）开发扩展的遗传字母表的体外应用。完成 这些目标应产生一个扩大的遗传字母表/密码， 生物医学效用，以及奠定基础，为一个活的有机体与半， 合成基因组 图1. d5SICS：dNaM 碱基对

The diversity of all living organisms is encoded within their DNA, where it is stably maintained through DNA replication, and then retrieved through transcription into RNA and translation into proteins. Thus, the diversity of life is limited by the four natural nucleotides that comprise DNA and RNA and the twenty natural amino acids that they encode. Drawn by the potential conceptual and practical ramifications, chemists and biologists have been fascinated by the idea of expanding the genetic alphabet (DNA and RNA) as well as the genetic code (proteins). This requires an unnatural base pair that is replicated within DNA, transcribed into RNA, and then able to direct the incorporation of an unnatural amino acid into a protein during translation. Expansion of the genetic alphabet/code would make available DNA, RNA, and proteins that are tailored to possess novel properties - just as these biopolymers are receiving increased attention for applications ranging from novel materials to therapeutics. The ability to synthesize or evolve these biopolymers with desired activities outside the scope encoded by their natural constituents promises to greatly increase their potential applications. Expansion of the genetic alphabet/code would also lay the foundation for the first semi-synthetic organism, able to store increased information in its DNA and retrieve it in the form of novel proteins. A survey of the literature demonstrates that the expansion of the genetic alphabet/code, at least in vitro, is currently limited by the absence of a functional unnatural base pair that is replicated and transcribed with sufficient efficiency and fidelity. In the previous funding period we developed the first unnatural base pair, d5SICS:dNaM, that is replicated and transcribed with efficiency and fidelity that is beginning to approach that of a natural base pair. Molecular recognition within this pair is based not on complementary hydrogen-bonding, as with the natural base pairs, but on complementary hydrophobic and packing forces, more similar to proteins. We also developed a polymerase selection system capable of tailoring the enzymes responsible for the replication of DNA to better recognize the unnatural base pair. With these tools in hand, we now propose to use a variety of synthetic and biological methods to: 1) Characterize structural determinants of d5SICS:dNaM stability, replication, and transcription; 2) Optimize d5SICS:dNaM for natural like replication and transcription; and 3) Develop in vitro applications of an expanded genetic alphabet. The completion of these aims should produce an expanded genetic alphabet/code with biophysical and biomedical utility, as well as lay the foundation for a living organism with a semi- synthetic genome. Figure 1. The d5SICS:dNaM base pair