Folding RNA: from Aids to the Human Genome

描述（由申请人提供）：RNA病毒引起了20世纪的三大流行病：流感、小儿麻痹症和艾滋病毒。尽管RNA在几乎所有疾病中都起着核心作用，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通过利用指导RNA折叠的相互作用的知识，从RNA分子的序列中可靠地预测其结构。研究结果也有助于合理设计治疗方法。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。对寡核苷酸的热力学和结构性质的研究将为结构预测奠定基础。特别的重点将放在稳定性的序列依赖和局部三维结构的内部回路。利用自由能最小化法预测二级结构的方法将在核磁共振约束下得到增强。核磁共振辅助二级结构预测（NMR assisted prediction of secondary structure， NAPSS）方法通过识别典型碱基对的螺旋结构，利用两个未分配光谱来限制折叠空间。这允许确定假结，这是很难用其他方法预测。假结通常具有重要的功能，因此是潜在的治疗靶点。提出了一种基于核磁共振化学位移进一步限制折叠空间的方法。NAPSS也是为三维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)在之前的资助期内发现的一个新的和新颖的内部环基序的完整溶液结构将由核磁共振确定，(2)与这种新结构平衡的不太稳定的结构也将被确定，(3)单链寡核苷酸四聚体的结构方面将由核磁共振确定，并与分子动力学模拟的预测进行比较。预测方法的力量将通过实验确定预测折叠成稳定二级结构的流感RNA区域的二级和三维结构来测试。初步结果表明，假结和发夹之间的构象转换可能调节剪接。短寡核苷酸和小分子的微阵列将用于发现针对已发现结构的化合物，并可能作为自然菌株和任何生物恐怖主义工程的治疗方法。一旦基因组测序完成，这些方法为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.