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.

作为总结RNA结构领域Stuart Le Grice（CCR）领域的各种成就的一种手段，我本人编辑了《 Methods Journal》的特别版，名为RNA结构确定的进步。该版本包括19个贡献，其中包括我小组的一项贡献，每个贡献描述了RNA结构预测和分析中使用的各种方法。 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纳米。这个问题非常全面，涵盖了RNA结构的当前状态。我们先前发现了萝卜皱纹病毒tRNA样转化增强剂（TCV TSS）的结构，使我们能够使用相对新的技术来理解当光学镊子应用分子结构时，以理解RNA的结构特性。本质上，将力施加到分子的5和3质量末端，然后对其进行监测。然后，力变化与结构特征相关。与安妮·西蒙（Anne Simon）合作的拉动实验与模拟转导的分子动力学相关，这使分子的展开事件可视化，这是拉动速度和施加力的函数。粗粒和显式溶剂技术被用于阐明结构特征。该技术提供了一种独特的方法来理解RNA结构和结构中发现的各种RNA图案的特征。寨卡病毒是世界上新兴的威胁。尽管主要在热带地区流行，但由于雌性埃德斯埃及蚊子，它似乎正在北半球更温和的气候。由于病毒可能影响胎儿发育，例如小头畸形。寨卡病毒是一种黄体病毒，与Flaviviridae家族中的登革热病毒以及其他病毒有关。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。正在构建一个小型素组，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相互作用进行了全面检查。 MySeq读取正在研究并与潜在相互作用的计算分析相关联。确定的患病率或缺乏率是为了更好地理解细胞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理解microRNA的处理以及它们如何与正常的细胞活性以及癌症等疾病相关。正在应用实验和计算方法来确定这些关系。从最初的结果弯曲或靶向Drosha茎中的结构弯曲或扭曲的结构似乎有助于产生替代形式的microRNA。结构预测和实验结果正在比较和相关。最近启动了与Esta Sterneck的实验室合作。她的实验室研究了使用体外细胞培养和体内小鼠模型系统的转录因子CCAAT/增强子结合蛋白Delta（CEBPD）研究涉及乳腺癌和胶质母细胞瘤肿瘤发生的细胞信号传导途径。使用转基因小鼠的乳腺癌模型，她的组表明CEBPD在乳腺肿瘤发生中表现出双重作用。一方面，CEBPD可防止肿瘤多样性，另一方面，CEBPD促进远处的肺转移。此外，CEBPD通过调节各种信号途径和茎，促进了与肿瘤转移和抗治疗耐药性有关的干性癌细胞，这些癌细胞已与肿瘤转移和治疗耐药性有关。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。作为与我们的纳米生物学项目联系起来的，我们的实验室正在开发RNAi Therapeutics通过战略性设计的RNA纳米结构作为其自身实体或与脂质载体结合的方法来击倒CEBPD mRNA的方法。由于需要坚固地生成各种长度的大量RNA，出于各种目的，已经建立了与转录专家Mikhail Kashlev进行的合作，以使用常见的酶，大肠杆菌RNA聚合酶来实现此目的。这种需求部分是由于建立了RNA生物学实验室，潜在的需求是一种治疗性以及NIH社区的现有要求。目前，从专门从事生产的公司订购时，规模的生产成本很高。当需要在RNA的特定位置中包括修改基础时，成本变得更加令人望而却步。通常，化学合成技术仅限于100个以下的碱基，并且是使用RNA T7聚合酶的常见方法，当需要修改碱时，对于某些序列而言，对于某些序列而言，这对于某些序列可能并不是很好。大肠杆菌RNA聚合酶的使用为各种需求的RNA生产提供了潜在的途径。应用此方法的初步实验看起来令人鼓舞。 RNA次级和3D结构的预测是一个难题，是一个困难而重要的问题，需要更好的算法。我们正在开发一组计算算法，以实现RNA中规范和更重要的非经典基对相互作用的预测。已编译了一个大型数据库，其中包含许多结构，包括非规范基本对相互作用。该算法显示出明显的效用，从而实现了二级结构水平上复杂基序的预测。然后将这些结果与RNA 3D结构生成程序结合使用，该程序可以预测结合复杂的非人类相互作用的3D RNA结构。这组算法也应用于多序列RNA纳米组件的预测。