Chemical catalysis in ribozymes

被称为核酶的催化活性核糖核酸的发现，重新定义了RNA在生物学中的作用。尽管最近在RNA结构测定方面取得了进展，但RNA介导的催化作用的详细机制仍然不清楚。与蛋白质酶相比，RNA只利用了四种不同的构件，可用的功能基团要有限得多。因此，问题就出现了，核酶如何才能达到令人印象深刻的催化反应速度。我们实验室的最新结果表明，核酶活性部位的内部静电场对RNA的催化潜力有很大贡献。我们将利用两个RNA系统进行拟议的研究，天然的Varkud卫星核酶和体外选择的孔雀石绿色结合RNA适配子，我们将其设计成原始催化剂。我们将在完整核酶的背景下研究VS核酶活性部位结构域的结构，并使用核磁共振光谱、紫外/可见光谱以及计算方法表征孔雀石绿衍生物在RNA结合口袋内外的化学反应。此外，我们还将描述使这些核酶能够催化化学反应的作用力和分子内相互作用。这项资助中描述的工作的长期目标是了解这些核酶中的分子结构和作用力是如何促进催化的。具体地说，我们在问：RNA催化中心的结构是什么？这些结构如何影响与催化有关的核苷酸的电荷分布和化学性质？静电力在RNA催化中有多重要？我们能从化学上相对简单的核酶中得出关于生物催化剂的一般机理结论吗？这些研究还将有助于为系统设计具有改变特异性的新核酶奠定基础，这些核酶可用作分子生物学、化学、纳米技术和医学的工具。

The discovery of catalytically active ribonucleic acids, known as ribozymes, redefined the role of RNA in biology. Despite the recent progress in RNA structure determination, the detailed mechanisms by which RNA mediated catalysis is achieved remain elusive. In comparison to protein enzymes, RNA utilizes only four different building blocks and has a much more limited set of functional groups available. Thus the question arises of how ribozymes can achieve their impressive catalytic acceleration of reaction rates. Recent results from our laboratory suggest that the internal electrostatic field within the ribozyme active sites significantly contributes to the catalytic potential of RNA. We will utilize two RNA systems for the proposed studies, the natural Varkud satelite ribozyme, and the in vitro selected malachite green binding RNA aptamer, which we engineered in to a primitive catalyst. We will study the structure of the VS ribozyme active site domain in the context of the complete ribozyme and characterize chemical reactions of malachite green derivatives inside and outside the RNA binding pocket using NMR spectroscopy, Ultraviolet/Visible spectroscopy, as well as computational approaches. Furthermore we will characterize the forces and intramolecular interactions that enable these ribozymes to catalyze chemical reactions. The long-term objectives of the work described in this grant are to understand how molecular structure and forces within these ribozymes contribute to catalysis. Specifically we are asking: What are the structures of the RNA catalytic centers? How do these structures affect the charge distribution and chemical properties of nucleotides that are involved in catalysis? How important are electrostatic forces in RNA catalysis? And can we draw general mechanistic conclusions about biological catalysts from the chemically relatively simple ribozymes? The studies will also help to lay the groundwork for a systematic design of new ribozymes with altered specificities that can be used as tools in molecular biology, chemistry, nanotechnology, and medicine.