RNA Chemistry and the RNA World

RNA在核糖体核心的存在，核苷酸辅助因子的广泛使用，以及RNA前体对DNA和一些氨基酸的合成表明，RNA在生命进化的早期曾经是主要的催化和遗传聚合物。这种“RNA世界”假设认为，RNA能够进行维持和进化“核糖生物”所需的所有反应。我实验室的长期目标之一是选择和进化一种RNA复制酶，能够在实验室中维持基于RNA的进化。这样的系统可以被称为“活的”，它将化学的简单性与生物学的复杂性结合在一起。确定产生RNA复制所需的最小复杂性，将以前所未有的方式告知早期地球从非生物世界向生物世界过渡所面临的困难。目标i和ii：迈向RNA复制酶：我们将使用传统的和基于乳剂的体外选择方法来分离高进步性的优质RNA聚合酶核酶。目前最先进的聚合酶核酶能够快速形成磷酸二酯键，但几乎无法适当地保持它延伸的引物-模板底物。克服这一核心问题有可能将聚合速率提高十倍或更多，并提供了在以小时为单位的时间尺度内将模板扩展数百个核苷酸的前景-如果实现，这是一个令人兴奋的前景！目标II：易位机制和均聚聚合酶：我们将选择聚合酶核酶，将聚(A)尾部逐渐结合到RNA引物的末端。这种聚合酶很有趣，因为长均聚物的过程合成需要一个易位机制，在核苷酸结合后，必须重置聚合酶复合物，使其看起来完全像初始状态，以便进行迭代聚合。这种机制最终可以证明在RNA复制过程中取代非模板链是非常有用的，因此在复制酶核酶的形成中起着重要作用。

The presence of RNA at the core of the ribosome, the extensive use of nucleotide cofactors, and the synthesis of DNA and some amino acids from RNA precursors suggests that RNA was once the dominant catalytic and genetic polymer early in the evolution of life. This 'RNA World' hypothesis assumes that RNA is capable of all the reactions required to maintain and evolve a 'ribo-organism'. One of my laboratories long term objectives is to select and evolve an RNA replicase ribozyme capable of sustaining RNA based evolution in the laboratory. Such a system could be called 'alive' and would marry the underlying simplicity of chemistry to the complexity of biology. Identifying the minimal complexity required to produce an RNA replicace would inform as never before about the difficulties faced on the early Earth in transitioning from an abiotic to biotic world. Aims I&II: Towards an RNA replicase ribozyme: We will use conventional and emulsion based in vitro selection methodologies to isolate superior RNA polymerase ribozymes that are highly processive. The current state of the art polymerase ribozyme is capable of rapid phosphodiester bond formation, but is nearly unable to appropriately hold the primer-template substrate it extends. Overcoming this central problem has the potential to increase polymerization rates by factors of ten or more and offers the prospect of extending a template by hundreds of nucleotides in a time scale measured in hours - an exciting prospect if realized! Aim II: Translocation mechanism & homopolymer polymerases: We will select for polymerase ribozymes that processively incorporate poly(A) tails onto the end of an RNA primer. Such polymerases are interesting as the processive synthesis of a long homopolymer requires a translocation mechanism that, after the incorporation of a nucleotide, must reset the polymerase complex to look exactly like the initial state so as to allow iterative polymerization. Such a mechanism ultimately could prove very useful in displacing a non-template strand during RNA replication and thus play an important role in developing a replicase ribozyme.