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) 和我本人编辑了《方法》杂志的特刊，题为“RNA 结构测定的进展”。该版本包括 19 篇贡献，其中一篇来自我的团队，每一篇都描述了 RNA 结构预测和分析中使用的各种方法。贡献的例子包括在特定位点标记 RNA 的方法的描述、小角度 X 射线散射和原子力显微镜的使用、SHAPE 的使用、羟基自由基足迹、FRET、适体开发、包括粗粒度模拟技术在内的计算方法、RNA 折叠和 3D 结构预测、RNA 基序数据库以及生成基于 RNA 的方法 纳米环。本期内容相当全面，涵盖了 RNA 结构的最新技术水平。我们之前对萝卜皱纹病毒 tRNA 样翻译增强子 (TCV TSS) 结构的发现，使我们能够在使用光镊将分子结构拉开时，使用相对较新的技术来了解 RNA 的结构特征。本质上，对分子的 5 和 3 个素端施加力，然后对其进行监测。然后将力的变化与结构特征相关联。与 Anne Simon 合作的拉动实验与模拟引导分子动力学相关联，这使得分子的展开事件可视化为拉力速度和所施加的力的函数。粗粒度和显式溶剂技术被用来阐明结构特征。该技术提供了一种独特的方法来理解 RNA 结构以及结构中发现的各种 RNA 基序的特征。寨卡病毒是世界上正在出现的威胁。尽管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的温带气候传播。由于该病毒可能影响胎儿发育，例如胎儿发育，已向孕妇发出警告。小头畸形。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关。由于我们最近与 R. Padmanabhan 的合作以及有关登革热病毒的出版物，我们合作确定了该病毒的结构特征，其中一些似乎与登革热结构相似。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所涉及的机制。我们还与 Shuo Gu 合作，对细胞中发现的潜在 RNA-RNA 相互作用进行全面检查。 MySeq 读数正在接受检查，并与潜在相互作用的计算分析相关联。正在确定其普遍性或缺乏性，以便更好地了解细胞 RNA 如何与其细胞环境相互作用。 Drosha 在细胞系统中的功能对于了解 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，我们正在努力了解 Drosha 靶向茎环结构与产生的 microRNA 同工型数量之间的关系。正在应用实验和计算方法来确定这些关系。从初步结果来看，目标 Drosha 茎中的弯曲或扭曲结构似乎促进了替代形式 microRNA 的产生。结构预测和实验结果正在进行比较和关联。最近启动了与 Esta Sterneck 实验室的合作。她的实验室利用体外细胞培养和体内小鼠模型系统研究参与乳腺癌和胶质母细胞瘤肿瘤发生的细胞信号传导途径，重点关注转录因子 CCAAT/增强子结合蛋白 delta (CEBPD)。她的团队利用乳腺癌转基因小鼠模型表明，CEBPD 在乳腺肿瘤发生中发挥双重作用。一方面，CEBPD 预防肿瘤多重性，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，促进乳腺和胶质母细胞瘤细胞中的干细胞样癌细胞，这些细胞与肿瘤转移和治疗耐药有关。此外，针对 CEBPD 信息的策略对于下调 CEBPD 介导的肿瘤进展信号传导是必要的。作为与我们纳米生物学项目的结合，我们的实验室正在开发 RNAi 疗法的方法，通过提供战略设计的 RNA 纳米结构作为其自身实体或与脂质载体结合来敲除 CEBPD mRNA。由于需要稳健地生产大量不同长度和不同目的的 RNA，我们与转录专家 Mikhail Kashlev 建立了合作，使用常见的酶——大肠杆菌 RNA 聚合酶来实现这一目的。这种需求的出现部分是由于 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，向专门生产的公司订购规模化生产成本相当高。当需要将修饰碱基包含在 RNA 内的特定位置时，成本变得更加高昂。通常，化学合成技术仅限于 100 个碱基以下，并且当需要修饰碱基时，使用 RNA T7 聚合酶的常用方法可能对某些序列有用，但并不适用于所有序列组合物。大肠杆菌 RNA 聚合酶的使用为稳健生产满足各种需求的 RNA 提供了潜在途径。应用这种方法的初步实验看起来令人鼓舞。包含非规范碱基对相互作用的 RNA 二级和 3D 结构的预测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，以预测 RNA 中的规范碱基对相互作用，更重要的是预测非规范碱基对相互作用。已经编译了一个大型数据库，其中包含多种结构，包括非规范碱基对相互作用。该算法显示出显着的实用性，能够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，从而能够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纳米组装体的预测。