Computer Analysis of Nucleic Acid Structure

要完全了解RNA分子的功能，需要了解其高阶结构(2D和3D)及其一级序列的特征。RNA结构对许多功能很重要，包括转录和翻译的调节、催化和蛋白质的跨膜运输。对这些功能的了解对于基础生物学以及生物检测和药物开发都很重要，这些药物可以干预这些分子发生病理功能的情况。这可能包括艾滋病毒等病毒或恶性细胞中的表达途径。 我们已经开发并继续改进一种使用遗传算法概念的RNA折叠技术。该算法最初是在大规模并行SIMD(16,384个处理器)MasPar上开发的，目前运行在并行MIMD超级计算机上。该算法可以在SGI辛烷单处理机、处理机、SGI/Cray Origin2000处理机和512Cray T3E处理机上运行。该算法具有很好的可伸缩性，能够在数十万个虚拟处理器上运行，给出了与种群变化相关的显著结构结果。我们能够预测RNA假结，并探索包含多种功能构象的折叠途径。此外，该算法还包含其他功能，如Boltzmann松弛技术、不同能量规则的选择、模拟顺序折叠和顺序处理的能力、螺旋茎的强制/建议和禁止嵌入以及折叠动态的实时可视化。 STRUCTURELAB是一个多相生物信息学RNA分析工作台，它允许使用一系列广泛的方法来进行RNA结构分析，并不断得到改进，以便能够产生RNA结构的精细3D原子坐标以及这些结构的可视化。此外，对一种新的交互式可视化方法STEM TRACE进行了进一步的改进和扩展。这种方法能够从系统发育的角度比较和分析多个序列RNA折叠，从而允许改进一系列序列的预测结构结果。此外，当它与遗传算法(见上文)一起使用时，它允许可视化折叠路径。还可以产生基序模式，以便可以探索RNA序列家族以寻找共同的结构元素。这些系统已经适应了我们实验室和NIH内外的其他环境，并可根据要求提供。 该系统已用于研究RNA折叠途径及其功能中间体，如PSTV类病毒和HOK/SOK质粒，两者都进入对其生命周期重要的中间状态；最近，对HIV二聚区的折叠途径进行了详细的研究。此外，还研究了柯萨奇B组病毒心脏毒力表型的遗传机制，包括转录抑制机制；单链突变的p53 DNA结构与毛细管电泳法的相关性；以及酶胸苷合成酶(TS)和TS mRNA之间的RNA/蛋白质结构相互作用。在后者中，实验表明，人类TS mRNA的翻译是由一种负的自我调节机制控制的，它本身就是合成的蛋白质。在人类结肠癌细胞中已经发现了这些RNA/蛋白质复合体的存在。 为了在原子水平上理解RNA的结构、折叠途径以及RNA-蛋白质相互作用的结构效应，人们利用分子力学和分子动力学模拟方法研究了RNA分子的一些结构元素。目前正在研究的结构基序除了胸苷合成酶(如上所述)外，还包括出现在嗜热嗜热菌30S核糖体亚基中心结构域晶体结构中的RNA四环和三向连接。实验确定，三向连接与S15核糖体蛋白之间的分子间相互作用启动了30S核糖体亚基的组装过程。然而，仍然需要揭示三向连接细节的动态图像，包括它与S15核糖体蛋白的相互作用，以及与这种蛋白质相互作用相关的原子水平上发生的构象变化。通过分子动力学模拟，我们对与S15结合相关的连接的构象转变获得了有意义的见解。 此外，还研究了与温度相关的四环变性和随后的折叠到原始晶体结构的分子动力学轨迹。分析了S15核糖体蛋白对四环的稳定作用。在这两种情况下，除了揭示新的原子水平细节外，这些区域的结构跃迁与热力学和生物化学实验得出的结果相对应。 我们也一直在建模并将分子动力学技术应用于HIV接吻环结构。这导致了对微妙的原子水平相互作用的理解，这些相互作用最终可能对病毒的生命周期非常重要。

A complete understanding of the function of RNA molecules requires a knowledge of the higher order structures (2D and 3D) as well as the characteristics of their primary sequence. RNA structure is important for many functions, including regulation of transcription and translation, catalysis, and transport of proteins across membranes. The understanding of these functions are important for basic biology as well as for bioassays and the development of drugs that can intervene in cases where pathological functionality of these molecules occurs. This may include viruses such as HIV or expression pathways in malignant cells. We have developed and continue to improve upon an RNA folding technique that uses concepts from genetic algorithms. The algorithm was originally developed on a massively parallel SIMD (16,384 processors) MasPar and currently runs on parallel MIMD supercomputers. The algorithm can run on a single processor of an SGI OCTANE, a 64 processor SGI/CRAY ORIGIN 2000 as well as a 512 processor CRAY T3E. The algorithm scales extremely well and is capable of running with hundreds of thousands of virtual processors giving significant structural results related to population variation. We are able to predict RNA pseudoknots and explore folding pathways that contain multiple functional conformations. In addition, the algorithm contains other features such as a Boltzmann relaxation technique, a choice of different energy rules, the ability to simulate sequential folding as well as sequential processing, forced/suggested and inhibited embedding of helical stems and the visualization of folding dynamics in real time. STRUCTURELAB, the heterogeneous bioinformatical RNA analysis workbench, which permits the use of a broad array of approaches for RNA structure analysis, has been continually enhanced to enable the production of refined 3D atomic coordinates of RNA structures along with the visualization of these structures. Also, a novel interactive visualization methodology, STEM TRACE, has been further improved and extended. This methodology enables the comparison and analysis of multiple sequence RNA folds from a phylogenetic point of view, thus allowing improvement of predicted structural results across a family of sequences. In addition, it permits the visualization of folding pathways when used in conjunction with the genetic algorithm (see above). It is also possible to produce motif patterns so that families of RNA sequences can be explored for common structural elements. These systems have been adapted to other environments inside and outside our laboratory and NIH and are available upon request. This system has been employed in studying RNA folding pathways and their functional intermediates as exemplified by the PSTV viroids and the hok/sok plasmid, both of which enter into intermediate states that are important for their life cycle; More recently the folding pathway of the HIV dimerization region has been studied in detail. Also studied are the genetic mechanisms of the viral cardiovirulence phenotype of the coxsackie B viruses which includes mechanisms for transcription attenuation; correlation of single stranded mutated p53 DNA structure and capillary electrophoresis; and the RNA/protein structural interactions between the enzyme thymidylate synthase (TS) and TS mRNA. In the latter, experiments have indicated that translation of human TS mRNA is controlled by an a negative autoregulatory mechanism with its own synthesized protein. The existence of these RNA/protein complexes have been found in human colon cancer cells. In order to understand RNA structures, folding pathways and the structural effects of RNA-Protein interactions at the atomic level, some structural elements of RNA molecules are being studied using molecular mechanics and molecular dynamics simulations. The structural motifs currently being studied, in addition to thymidylate synthase (mentioned above), include RNA tetraloops and three-way junctions that appear in the crystal structure of the central domain of the 30S ribosomal subunit from Thermus thermophilus. It has been experimentally determined that the intermolecular interactions between the three-way junction and the S15 ribosomal protein initiate the process of the assembly of the 30S ribosomal subunit. It remains however, to reveal the dynamic picture of the details of the three-way junction including its interaction with the S15 ribosomal protein and the conformational changes that take place at the atomic level associated with this protein interaction. By using molecular dynamics simulations we have obtained significant insights into the conformational transitions of the junction associated with the binding of S15. In addition, the molecular dynamics trajectories associated with temperature dependent denaturation of a tetra-loop and the subsequent refolding to the original crystal structure have been examined. The stablizing influence of the S15 ribosomal protein on the tetra-loop has been analyzed. In both of these cases, besides revealing new atomic level details, the structural transitions in these regions correspond to results derived from thermodynamic and biochemical experiments. We have also been modelling and applying molecular dynamics techniques to the HIV kissing loop structure. This has lead to the understanding of subtle atomic level interactions that may ultimately be quite significant to the viral life cycle.