Folding RNA: from Aids to the Human Genome

描述（由申请人提供）：RNA病毒引起了世纪的三大流行病，流感、脊髓灰质炎和HIV。尽管RNA在基本上所有疾病中起着核心作用，但可能成为治疗靶点的RNA结构在很大程度上是未知的。本研究的最终目标是利用指导RNA折叠的相互作用的知识，从RNA分子的序列中可靠地预测RNA分子的结构。研究结果还将引导合理设计治疗方法。预测RNA结构的能力也有助于解释由基因组计划和其他测序工作确定的序列。 通过对寡核苷酸热力学和结构性质的研究，为结构预测奠定了基础。特别强调将放在序列的稳定性和局部三维结构的内部循环的依赖。通过自由能最小化的二级结构预测将增加NMR的约束。NMR辅助二级结构预测（NAPSS）方法使用两个未分配的光谱，通过识别典型碱基对的螺旋来限制折叠空间。这允许确定伪结，这是难以预测的其他方法。假结通常具有重要的功能，因此是潜在的治疗靶点。提出了一种方法，进一步限制折叠空间的基础上NMR化学位移。NAPSS也是为3D结构确定分配共振的第一步。 为了推进3D结构的预测，将开发基准来测试力场和计算方法。例如，（1）在前一个资助期发现的新的和新颖的内环基序的完整溶液结构将通过NMR确定，（2）与这种新结构平衡的不太稳定的结构也将被确定，以及（3）单链寡核糖核苷酸四聚体的结构方面将通过NMR确定，并与分子动力学模拟的预测进行比较。 预测方法的能力将通过实验确定预测折叠成稳定二级结构的流感RNA区域的二级和3D结构来测试。初步结果表明，有一个假结和发夹之间的构象开关，可以调节剪接。短寡核苷酸和小分子的微阵列将用于发现靶向所发现结构的化合物，并可能作为天然菌株和任何生物恐怖主义工程的治疗剂。这些方法为一旦基因组测序后快速设计治疗方法提供了基础。

DESCRIPTION (provided by applicant): RNA viruses caused the three major pandemics of the 20th century, influenza, polio, and HIV. Despite the central roles of RNA in essentially all diseases, the RNA structures that can potentially be therapeutic targets are largely unknown. The ultimate goal of this research is to predict reliably the structures of RNA molecules from their sequences by using knowledge of the interactions directing RNA folding. The results should also lead to rational design of therapeutics. The ability to predict RNA structure also facilitates interpretation of sequences determined by genome projects and other sequencing efforts. The foundation for structure prediction will be advanced by studies of the thermodynamic and structural properties of oligonucleotides. Particular emphasis will be placed on the sequence dependence of stability and local three dimensional structures of internal loops. Secondary structure prediction by free energy minimization will be augmented with constraints from NMR. The NMR assisted prediction of secondary structure (NAPSS) method uses two unassigned spectra to restrict folding space by identifying helixes of canonical base pairs. This allows determination of pseudoknots, which are difficult to predict with other methods. Pseudoknots usually have significant functions, and are thus potential therapeutic targets. An approach is proposed that restricts folding space further on the basis of NMR chemical shifts. NAPSS is also a first step in assigning resonances for 3D structure determination. To advance predictions of 3D structure, benchmarks will be developed to test force fields and computational methods. For example, (1) the complete solution structure of a new and novel internal loop motif discovered in the previous grant period will be determined by NMR, (2) a less stable structure in equilibrium with this novel structure will also be determined, and (3) structural aspects of single stranded oligoribonucleotide tetramers will be determined by NMR and compared to predictions from molecular dynamics simulations. The power of predictive methods will be tested by experimentally determining the secondary and 3D structures of regions of influenza RNA predicted to fold into stable secondary structures. Initial results indicate there is a conformational switch between a pseudoknot and hairpin that may regulate splicing. Microarrays of short oligonucleotides and small molecules will be used to discover compounds that target the discovered structures and could potentially serve as therapeutics for natural strains and any engineered for bioterrorism. These approaches provide the foundation for rapidly designing therapeutics once a genome has been sequenced.