Computational Approaches for RNA StructureFunction Determination

RNA 结构功能测定的计算方法

• 批准号: 9556215
• 负责人: Bruce Shapiro
• 金额: $ 48.24万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556215
• 关键词: Aedes; Affect; Algorithms; Atomic Force Microscopy; Base Pairing; Biological; Biological Assay; Biological Models; Biology; Breast; Breast Cancer Model; CCAAT-enhancer-binding protein-delta; CCR; Carrier Proteins; Catalysis; Categories; Cell Culture Techniques; Cell physiology; Cells; Cereals; Characteristics; Climate; Collaborations; Communities; Complex; Computational algorithm; Computer Analysis; Computer software; Computing Methodologies; Culicidae; DNA-Directed RNA Polymerase; Databases; Dengue; Dengue Virus; Development; Dimensions; Disease; Distant; Enhancers; Environment; Enzymes; Escherichia coli; Event; Exhibits; Family; Female; Fetal Development; Flavivirus; Fluorescence Resonance Energy Transfer; Gene Silencing; Generations; Genetic Transcription; Glioblastoma; Higher Order Chromatin Structure; Hydroxyl Radical; Imagery; In Vitro; Journals; Knowledge; Label; Laboratories; Length; Lipids; Malignant Neoplasms; Mammary Tumorigenesis; Mediating; Membrane; Messenger RNA; Metastatic Neoplasm to the Lung; Methodology; Methods; MicroRNAs; Microcephaly; Molecular Structure; Monitor; Nanostructures; Neoplasm Metastasis; Pathologic; Pathway interactions; Positioning Attribute; Pregnant Women; Prevalence; Production; Publications; RNA; RNA Databases; RNA Folding; RNA Interference; RNA Sequences; RNA analysis; Regulation; Research; Roentgen Rays; Role; Signal Pathway; Signal Transduction; Site; Solvents; Speed; Structure; Structure-Activity Relationship; System; T7 RNA polymerase; Techniques; Therapeutic; Transcriptional Regulation; Transfer RNA; Transgenic Mice; Translations; Turnip - dietary; United States National Institutes of Health; Viral; Virus; Virus Replication; Zika Virus; aptamer; base; cancer cell; chemical synthesis; cost; design; drug development; experimental study; in vivo; knock-down; laser tweezer; molecular dynamics; mouse model; nanoassembly; nanobiology; neoplastic cell; prevent; programs; protein transport; simulation; stem; stemness; therapy resistant; three dimensional structure; transcription factor; tumor; tumor progression; tumorigenesis; virus characteristic; web site

As a means to summarize various accomplishments in the field of RNA structure Stuart Le Grice (CCR) and myself edited a special edition of the Methods journal entitled Advances in RNA Structure Determination. The edition included 19 contributions, including one from my group, each describing various methodologies used in RNA structure prediction and analysis. Examples of contributions included descriptions of methods for labeling RNAs at specific sites, the use of small angle x-ray scattering and atomic force microscopy, the use of SHAPE, hydroxyl radical footprinting, FRET, aptamer development, computational methodologies including coarse-grained simulation techniques, RNA folding and 3D structure prediction, a database of RNA motifs and a method for generating RNA-based nanorings. The issue is quite comprehensive, covering the current state of the art of RNA structure. Our previous discovery of the structure of the turnip crinkle virus tRNA-like translational enhancer (TCV TSS) has permitted us to pursue the use of a relatively new technique for understanding the structural characteristics of an RNA when optical tweezers are applied to pull the molecular structure apart. Essentially a force is applied to the 5 and 3 prime ends of the molecule, which is then monitored. Force changes are then correlated with structural features. The pulling experiments, in collaboration with Anne Simon, are being correlated to simulated steered molecular dynamics, which enables the visualization of the unfolding events of the molecule as a function of the pulling speed and forces applied. Coarse-grained and explicit solvent techniques are being used to elucidate the structural characteristics. This technique offers a unique methodology for understanding RNA structure and the characteristics of various RNA motifs found in the structure. The Zika virus is an emerging threat in the world. Although mostly prevalent in tropical zones it appears to be spreading to more temperate climates in the northern hemisphere due to the female Aedes aegypti mosquito. Warnings have been issued to pregnant women due to the potential for the virus to affect fetal development e.g. microcephaly. Zika virus is a Flavivirus and is related to the dengue viruses as well as other viruses in the Flaviviridae family. Due to our recent collaborations with R. Padmanabhan, and publications on the dengue virus, we collaborating on determining the structural characteristics of the virus some of which appear to be similar to the dengue structure. A minigenome is being constructed to further elucidate the mechanisms involved in Zika viral replication and translation. We are also pursuing, in collaboration with Shuo Gu, a comprehensive examination of potential RNA-RNA interactions that are found in cells. MySeq reads are being examined and correlated with computational analysis of potential interactions. The prevalence or lack thereof is being determined to enable a better understanding of how cellular RNA interacts with its cellular environment. The functionality of Drosha in cellular systems is important for understanding the processing of microRNAs and how they relate to normal cellular activity as well as diseases such as cancer.In another collaboration with Shuo Gu we are working on understanding the relationship of Drosha targeted stem-loop structures and the number of microRNA isforms that are produced. Experimental and computational approaches are being applied to determine these relationships. From initial results bent or distorted structures in the targeted Drosha stem seem to facilitate the production of alternate forms of microRNA. Structural predictions and experimental results are being compared and correlated. A collaboration with Esta Sterneck's laboratory was recently initiated. Her lab investigates cell signaling pathways involved in breast and glioblastoma tumorigenesis with a focus on the transcription factor CCAAT/enhancer binding protein delta (CEBPD) using in vitro cell culture and in vivo mouse model systems. Using a transgenic mouse model of breast cancer, Her group has shown that CEBPD exhibits a dual role in mammary tumorigenesis. On the one hand, CEBPD prevents tumor multiplicity and on the other hand, CEBPD promotes distant lung metastases. In addition, CEBPD promotes stem-like cancer cells, which have been implicated in tumor metastasis and treatment resistance, in breast and glioblastoma tumor cells through regulation of various signaling pathways and stemness. In addition, strategies for targeting the message of CEBPD are necessary to downregulate CEBPD-mediated tumor progression signaling. As a tie in to our nanobiology project, our laboratory is developing approaches for RNAi therapeutics to knock down the CEBPD mRNA by delivering strategically designed RNA nanostructures as their own entities or in combination with lipid carriers. Due to the need to robustly produce large quantities of RNA of various lengths and for various purposes, a collaboration with Mikhail Kashlev, an expert in transcription, has been established to accomplish this purpose using a common enzyme, E. coli RNA polymerase. This need has arisen, in part, due to the establishment of the RNA Biology Laboratory, potential needs as a therapeutic, as well as existing requirements of the NIH community. Currently, scaled production costs are quite high when ordering from companies that specialize in production. Costs become even more prohibitive when modified bases need to be included at specific positions within the RNA. Typically, chemical synthesis techniques are limited to under 100 bases and a common method of using RNA T7 polymerase, which may be useful for certain sequences does not perform well for all sequence compositions when modified bases are required. The use of E. coli RNA polymerase provides a potential avenue for the robust production of RNA for a variety of needs. Initial experiments applying this methodology look encouraging. The prediction of RNA secondary and 3D structures containing non-canonical base pair interactions is a difficult and important problem that needs better algorithms. We are developing a set of computational algorithms to enable the prediction of canonical and more importantly non-canonical base pair interactions in RNA. A large database has been compiled containing a multitude of structures including the non-canonical base pair interactions. The algorithms have shown significant utility, enabling the prediction of complex motifs at the secondary structure level. These results are then being used in conjunction with an RNA 3D structure generation program, which enables the prediction of 3D RNA structures that incorporate the complex non-canonical interactions. This set of algorithms are also being applied to the prediction of multi-sequence RNA nano-assemblies.

为了总结RNA结构领域的各种成就，Stuart Le Grice （CCR）和我编辑了《方法》杂志的特别版，题为《RNA结构测定的进展》。该版本包括19篇文章，其中一篇来自我的小组，每一篇都描述了用于RNA结构预测和分析的各种方法。贡献的例子包括描述在特定位点标记RNA的方法，使用小角度x射线散射和原子力显微镜，使用SHAPE，羟基自由基足迹，FRET，适体开发，计算方法包括粗粒度模拟技术，RNA折叠和3D结构预测，RNA基序数据库和生成基于RNA的纳米结构的方法。这个问题是相当全面的，涵盖了RNA结构的当前艺术状态。我们之前对萝卜皱病毒trna样翻译增强子（TCV TSS）结构的发现，使我们能够使用一种相对较新的技术来理解RNA的结构特征，当使用光学镊子将分子结构分开时。本质上，一个力被施加到分子的5和3 '端，然后被监测。然后，力的变化与结构特征相关联。与安妮·西蒙合作进行的拉扯实验与模拟操纵分子动力学相关联，这使得分子展开事件作为拉扯速度和施加的力的函数变得可视化。粗粒度和显式溶剂技术被用来阐明其结构特征。这项技术为理解RNA结构和结构中发现的各种RNA基序的特征提供了一种独特的方法。寨卡病毒是全球新出现的威胁。虽然主要流行于热带地区，但由于雌性埃及伊蚊，它似乎正在向北半球更温和的气候蔓延。由于该病毒可能影响胎儿发育，例如小头畸形，已向孕妇发出警告。寨卡病毒是一种黄病毒，与登革热病毒以及黄病毒科的其他病毒有亲缘关系。由于我们最近与R. Padmanabhan的合作，以及关于登革热病毒的出版物，我们合作确定病毒的结构特征，其中一些似乎与登革热结构相似。目前正在构建一个小基因组，以进一步阐明寨卡病毒复制和翻译的机制。我们还在与顾硕合作，对细胞中潜在的RNA-RNA相互作用进行全面研究。正在检查MySeq读数，并与潜在相互作用的计算分析相关联。为了更好地理解细胞RNA如何与其细胞环境相互作用，正在确定其流行与否。Drosha在细胞系统中的功能对于理解microrna的加工及其与正常细胞活动以及癌症等疾病的关系非常重要。在与Shuo Gu的另一项合作中，我们正在研究Drosha靶向茎环结构与产生的microRNA异构体数量之间的关系。正在应用实验和计算方法来确定这些关系。从最初的结果来看，在目标Drosha茎中弯曲或扭曲的结构似乎促进了microRNA替代形式的产生。结构预测和实验结果正在进行比较和关联。最近开始与Esta Sterneck的实验室合作。她的实验室利用体外细胞培养和体内小鼠模型系统研究了参与乳腺和胶质母细胞瘤肿瘤发生的细胞信号通路，重点研究了转录因子CCAAT/增强子结合蛋白δ (CEBPD)。利用转基因乳腺癌小鼠模型，她的研究小组发现CEBPD在乳腺肿瘤发生中具有双重作用。一方面，CEBPD抑制肿瘤的多样性，另一方面，CEBPD促进远处肺转移。此外，CEBPD通过调节各种信号通路和干性，促进乳腺癌和胶质母细胞瘤细胞中的干细胞样癌细胞，干细胞样癌细胞与肿瘤转移和治疗耐药有关。此外，为了下调CEBPD介导的肿瘤进展信号，需要靶向CEBPD信息的策略。作为我们纳米生物学项目的一部分，我们的实验室正在开发RNAi疗法的方法，通过递送策略设计的RNA纳米结构作为它们自己的实体或与脂质载体结合来敲除CEBPD mRNA。由于需要大量生产各种长度和各种用途的RNA，我们与转录专家Mikhail Kashlev合作，利用一种常见的酶——大肠杆菌RNA聚合酶来实现这一目的。这种需求已经出现，部分原因是由于RNA生物学实验室的建立，作为治疗的潜在需求，以及NIH社区的现有要求。目前，从专门生产的公司订购时，规模生产成本相当高。当需要将修饰的碱基包含在RNA内的特定位置时，成本变得更加高昂。通常，化学合成技术被限制在100个碱基以下，使用RNA T7聚合酶的常用方法可能对某些序列有用，但当需要修饰碱基时，对所有序列组成都表现不佳。大肠杆菌RNA聚合酶的使用为大量生产满足多种需求的RNA提供了一条潜在途径。应用这种方法的初步实验看起来令人鼓舞。预测含有非规范碱基对相互作用的RNA二级结构和三维结构是一个困难而重要的问题，需要更好的算法。我们正在开发一套计算算法来预测RNA中规范和更重要的非规范碱基对相互作用。已经编译了一个包含大量结构的大型数据库，其中包括非规范碱基对相互作用。这些算法已经显示出显著的实用性，可以在二级结构水平上预测复杂的基序。然后将这些结果与RNA 3D结构生成程序结合使用，该程序可以预测包含复杂非规范相互作用的3D RNA结构。这套算法也被应用于多序列RNA纳米组装的预测。