ANALYSIS OF CONSERVED 3D RNA MOTIFS

生物活性RNA分子分层折叠形成复杂的三维结构。 RNA分子的折叠状态通过碱基间堆叠相互作用和边对边碱基配对来稳定，由静电驱动的氢键相互作用介导。在前一个资助期内，对RNA中的碱基配对进行了详尽的分析。提出了一个系统的命名几何考虑的基础上，明确定义每个碱基配对的几何形状，并很容易确定等排相关的对。这种方法的价值，确定经常性的RNA基序的基础上，在同源RNA分子的序列比较证明。典型RNA碱基对的等排性是二级结构测定的比较方法的基础。该提案的目标是通过确定在高分辨率结构中鉴定的每个等排配对几何结构的等排矩阵，将该方法扩展到非规范碱基对，建立在前一个资助期的结果的基础上。将根据系统发育序列变异分析结构数据库（NDB和PDB）中可用的实验确定的三维RNA结构。潜在的电子等排碱基取代将通过在实验结构中手动建模和使用具有明确溶剂分子和抗衡离子的AMBER力场进行纳秒分子动力学模拟以及精确计算静电相互作用的粒子网格埃瓦尔德（PME）方法进行评估。将选定的碱基取代掺入合成的寡核苷酸中，并测量其对热解链行为的影响，以提供独立的实验数据，与计算机建模和模拟进行比较。还将进行RNA中碱基-碱基堆积模式的系统分析。初步数据表明，堆叠模式是依赖于序列和经常性的。在不同的背景下，对于特定的序列（例如5 '-AC-3'或5-UA-3 '），可以看到可重叠的堆叠模式。此外，某些序列表现出比其他序列更大的链内或跨链堆叠倾向。 首先通过检查所有可用结构中每个二核苷酸的所有出现情况来系统地分析链内堆积。提案中列举了这一办法已经取得的成果的例子。然后分析链间堆叠。链内堆积倾向是特别感兴趣的，因为局部堆积碱基的瞬时形成似乎在RNA折叠的早期阶段成核中发挥重要作用，例如在稳定发夹环的形成中。

Biologically active RNA molecules fold hierarchically to form complex 3- dimensional structures. Folded states of RNA molecules are stabilized by base-upon-base stacking interactions and edge-to-edge, base-pairing, mediated by electrostatically driven hydrogen bonding interactions. In the previous grant period an exhaustive analysis of base-pairing in RNA was carried out. A systematic nomenclature based on geometric considerations was proposed which serves to unambiguously define each base-pairing geometry and to readily identify isosterically related pairs. The value of this approach for identifying recurrent RNA motifs on the basis of sequence comparisons in homologous RNA molecules was demonstrated. The isostericity of the canonical RNA basepairs is the basis of the comparative approach to secondary structure determination. The goal of this proposal is to extend this approach to non-canonical base pairs by determining Isostericity Matricies for each isosteric pairing geometry identified in high- resolution structures, building on the results of the previous grant period. Experimentally determined three-dimensional RNA structures available in the structure databases (NDB and PDB) will be analyzed in light of phylogenetic sequence variations. Potentially isosteric base substitutions will be evaluated by hand-modeling in the experimental structures and by carrying out nano-second molecular dynamics simulations using the AMBER force field with explicit solvent molecules and counterions, and the particle mesh Ewald (PME) method to accurately calculate electrostatic interactions. Selected base substitutions will be incorporated into synthetic oligonucleotides, and their effect on the thermal melting behavior will be measured to provide independent experimental data to compare with computer modeling and simulation. The systematic analysis of base-base stacking patterns in RNA will also be undertaken. Preliminary data indicate that stacking patterns are sequence-dependent and recurrent. Superimposable stacking patterns are seen for particular sequences (for example 5'-AC-3' or 5-UA-3') in different contexts. Moreover, certain sequences exhibit a greater propensity for intra- or cross-strand stacking than others. Intra-strand stacking will first be systematically analyzed by examining all occurrences of each dinucleotide in all available structures. Examples of results this approach has already yielded are presented in the proposal. Inter-strand stacking will then be analyzed. Intra-strand stacking propensities are of particular interest as transient formation of locally stacked bases appears to play an important role in nucleating the early stages of RNA folding, for example in the formation of stable hairpin loops.