Mechanistic studies and engineering of reverse nucleotide polymerization

在自然界中，DNA和RNA聚合酶在细胞内完成许多基本的生物学功能，如复制遗传信息并将其转录成信使RNA(MRNA)。然后，这些mRNA被解码成蛋白质，这些蛋白质执行许多维持生命的必要活动。DNA和RNA复制蛋白(称为聚合酶)已经被用于技术目的，现在被广泛用于生物医学研究和生物技术工业。它们的一些应用包括全基因组测序、细菌和病毒感染的检测、亲子鉴定和法医学。自从聚合酶商业化以来，分子生物学已经发生了革命性的变化。据估计，仅一个聚合酶(Taq DNA聚合酶)的专利就产生了超过20亿美元的版税。所有已知的和商业上可用的聚合酶都是以“正向”方向唯一地复制DNA或RNA。这些聚合酶使用现有的模板链作为蓝图来创建互补链，这与从照片底片图像生成照片相媲美。相反的过程(即从照片中捕获照片底片)本质上是一个必要但非常复杂的过程，因为没有已知的聚合酶能够以相反的方向复制。这种“反向聚合酶”将有许多生物医学和生物技术应用，例如生物医学成像中的RNA和DNA标记，以及对通常“不可读”的上游序列进行测序。反向聚合酶将成为世界各地分子生物学实验室的重要工具，也将在癌症筛查和诊断中有重要的临床应用。我发现反向聚合确实是可能的。令人惊讶的是，一种小蛋白(tRNAHis鸟苷酸转移酶，Thg1)能够进行反向核苷酸加成。Thg1使用与正向聚合酶相同的机制、结构和催化特性。Thg1已经包含了反向聚合酶所需的所有成分，包括催化棕榈区(复制机)和定位核苷酸模板以进行反向复制的手段。最重要的是，我发现某些Thg1变种能够催化反向复制反应。在自然界中，Thg1仅限于一种特定的功能，但这种独特的酶有可能被改造成执行各种新的活动。我将利用Thg1已经执行反向核苷酸加成的事实来设计新的聚合酶，并将其应用于活细胞中的RNA和DNA标记。拟议的研究将推动聚合酶研究领域的发展，因为它将展示聚合如何在正向和反向进行。所获得的知识将导致可用于延长反向聚合的新酶，这些酶可用于克服目前分子生物学和生物医学成像中存在的生物技术挑战。

In nature, DNA and RNA polymerases fulfill numerous essential biological functions within the cell, such as copying genetic information and transcribing it into messenger RNA (mRNA). The mRNAs are then decoded into proteins that perform many of the essential activities to sustain life. DNA and RNA copying proteins (known as polymerases) have been harnessed for technological purposes and are now widely used in biomedical research and in the biotechnology industry. Some of their applications include the sequencing of whole genomes, detection of bacterial and viral infections, paternity testing, and forensics. Molecular biology has been revolutionized since polymerases became commercially available. The patent on just one polymerase (Taq DNA polymerase) is estimated to have generated over $2 billion in royalties. All known and commercially available polymerases copy DNA or RNA exclusively in the “forward” direction. These polymerases use an existing template strand as the blueprint to create a complementary strand, which is comparable to the generation of a photograph from a photo negative image. The reverse process (i.e., capturing the photo negative from a photograph) is a necessary but highly complicated process in nature, since no known polymerase is able to copy in the "reverse" direction. Such a “reverse polymerase” would have many biomedical and biotechnological applications, such as RNA and DNA labeling in biomedical imaging, and sequencing of normally “unreadable” upstream sequences. A reverse polymerase will become an essential tool in molecular biology laboratories around the world, and will also have important clinical applications in cancer screening and diagnosis.I discovered that reverse polymerization is indeed possible. It came as a surprise that a small protein (tRNAHis guanylyltransferase, Thg1) is capable of reverse nucleotide addition. Thg1 uses the same mechanistic, structural, and catalytic features as the forward polymerases. Thg1 already contains all the components a reverse polymerase requires, including a catalytic palm domain (copying machine) and the means of positioning the nucleotide template for reverse copying. Most importantly, I discovered that certain Thg1 variants are able to catalyze the reverse copying reaction. In nature, Thg1 is limited to a specific function, yet this unique enzyme has the potential to be engineered to carry out a variety of novel activities. I will exploit the fact that Thg1 already executes reverse nucleotide addition to engineer new polymerases with applications in RNA and DNA labeling in living cells. The proposed studies will advance the field of polymerase research, as it will show how polymerization can proceed in both forward and reverse direction. The knowledge gained will lead to new enzymes for extended reverse polymerization that can be applied to overcome biotechnological challenges currently present in molecular biology and biomedical imaging.