CAREER: Investigations of RNA Folding: A Combinatorial Approach to Thermodynamics

bevilacqua核酸（NA）是由A、C、G和T（或U）碱基组成的聚合物，它们像串珠一样以线性方式连接在一起。根据单个氧原子或甲基（CH3）基团的细微差别，自然产生的核酸可以正式分为DNA和RNA两种类型。生命的两个基本功能是储存决定个体身份的遗传信息，以及增加生命所必需的化学反应的速率和特异性，例如新陈代谢和复制。在细胞中，DNA被用来储存遗传信息，而蛋白质则像酶一样加速反应速率。引人注目的是，RNA既可以储存遗传信息，如引起艾滋病的HIV-1等某些病毒所证明的那样，又可以作为一种酶，RNA酶被称为“核酶”。这些特性使RNA参与了各种各样的生物过程和疾病，使RNA的结构和功能备受关注。此外，RNA被认为是益生元化学和生命起源的重要分子，因为RNA有可能解决“先有鸡还是先有蛋的问题”，是酶还是遗传信息。RNA的功能特性源于广泛的复杂三维结构。本提案中描述的研究的主要目标是了解RNA如何折叠成这些结构。RNA的序列，或四种碱基发生的顺序，可以很容易地得到。例如，已经完成了大约25种不同生物的基因组测序，预计将在未来五年内完成30亿个DNA碱基的人类基因组测序。为了了解这些rna的功能，结构模型是必不可少的，而预测结构的计算机方法对于跟上测序项目的步伐至关重要。PI的实验室正致力于用一种新颖的方法获得计算机预测的规则。研究人员正在开发一种组合方法，在这种方法中，根据热力学稳定性或结构对热变性的抵抗力，制备了一组含有多达一百万亿个不同序列的分子，然后在一次实验中分离出来。分离是在含有温度梯度（TGGE）的凝胶电泳装置上进行的。特别稳定和不稳定的序列将通过克隆和测序分离和鉴定，并通过对所选序列的进一步物理表征获得结构预测规则。这些方法将被应用于核酸中广泛的结构基序。这项研究将有助于更好地理解RNA折叠的基本原理。

9984129Bevilacqua Nucleic acids (NA) are polymers consisting of four building blocks--A, C, G, and T (or U) bases--attached in a linear fashion like beads on a string. Naturally occurring nucleic acids can be formally divided into two types, DNA and RNA, based on subtle differences of a single oxygen atom or methyl (CH3) group. Two fundamental functions essential to life are the storage of genetic information that determines an individual's identity, and the increase in the rate and specificity of chemical reactions essential to life, for example in metabolism and replication. In the cell, DNA is used to store genetic information, while proteins act as enzymes to accelerate reaction rates. Strikingly, RNA can both store genetic information, as evidenced by certain viruses such as HIV-1 which causes AIDS, and act as an enzyme, with RNA enzymes referred to as "ribozymes". These properties implicate RNA in a wide variety of biological processes and diseases, making the structures and functions of RNA of significant interest. Furthermore, RNA has been implicated as an important molecule in prebiotic chemistry and the origin of life, since RNA potentially solves the "chicken and egg problem" of which came first, enzymes or genetic information. The functional properties of RNA arise from a wide range of complex three-dimensional structures. Research described in this proposal has as its primary goal understanding how RNA folds into these structures. The sequences of RNA, or the order in which the four bases occur, can be readily obtained. For example, sequencing of the genomes of approximately 25 different organisms has been completed, and sequencing of the human genome with 3 billion DNA bases is expected to be completed in the next five years. To understand the function of these RNAs, structural models are indispensable, and computer methods to predict structures are essential for keeping pace with sequencing projects. The PI's laboratory is working to obtain rules for computer prediction using a novel methodology. Investigators are developing a combinatorial approach in which a collection of molecules containing up to a million billion different sequences are prepared and then separated in a single experiment according to thermodynamic stability, or the resistance of the structure to heat denaturation. Separations are on a gel electrophoresis apparatus containing a temperature gradient (TGGE). Exceptionally stable and unstable sequences will be isolated and identified by cloning and sequencing, and rules for structure prediction obtained by further physical characterization of selected sequences. These methods will be applied to a wide range of structural motifs in nucleic acids. The proposed research should provide a better understanding of basic principles of RNA folding.