Computational RNA Nanodesign

计算RNA纳米设计

• 批准号: 8349306
• 负责人: Bruce Shapiro
• 金额: $ 86.09万
• 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/8349306
• 关键词: Academia; Automation; B-DNA; Biochemical; Biocompatible Materials; Biological Assay; Caliber; Cereals; Chemicals; Chemistry; China; Collaborations; Complex; Computer Simulation; Computing Methodologies; DNA; Data; Databases; Detection; Development; Dicer Enzyme; Dimensions; Discipline; Disease; Drug Industry; Electron Microscopy; Endotoxins; Engineering; Environment; France; Functional RNA; Gel; Genetic; Genetic Transcription; Goals; Government; HIV; Human; Hybrids; In Vitro; International; Knowledge; Label; Major Groove; Malignant Neoplasms; Methodology; Methods; Minor Groove; Modeling; Modification; Molecular; Molecular Conformation; Motion; Nanostructures; Nanotechnology; Normal Range; Nucleotides; Oligonucleotides; Pathway interactions; Polymers; Principal Component Analysis; Process; Production; Property; Protein Binding; Protocols documentation; RNA; RNA Conformation; RNA I; RNA Interference; RNA chemical synthesis; Receptor Cell; Recombinants; Reporting; Research Personnel; Resistance; Ribonucleases; Route; Series; Shapes; Small Interfering RNA; South Korea; Specific qualifier value; Structure; Sweden; System; Technology; Testing; Therapeutic; Time; United States; Variant; Vertebral column; Vision; analog; aptamer; base; catalyst; chemical synthesis; cryogenics; design; flexibility; human DICER1 protein; interest; light scattering; meetings; molecular dynamics; nano; nanobiology; nanobiotechnology; nanodevice; nanomedicine; nanoparticle; nanoscale; nanosecond; particle; pre-clinical; programs; scaffold; self assembly; sensor; simulation; single molecule; stem; sugar; symposium; synthetic biology

In Vitro Assembly of Cubic RNA-based Scaffolds Designed in Silico The organization of biological materials into versatile three dimensional assemblies could be used to build multifunctional therapeutic scaffolds for use in nanomedicine. We reported a strategy to computationally design and experimentally verify the formation of three-dimensional cubic nanoscale scaffolds that can self-assemble from RNA (also DNA and RNA/DNA hybrids) with precise control over their shape, size and composition. These cubic nanoscaffolds are only 13 nm in diameter and are composed of short oligonucleotides, making them amenable to chemical synthesis, point modifications and further functionalization. Nanocube assembly was verified by gel assays, dynamic light scattering and cryogenic electron microscopy. Formation of functional RNA nanocubes was also demonstrated by the use of a fluorescent RNA aptamer that was optimally active only upon full RNA assembly. We showed that the RNA nanoscaffolds can self-assemble in isothermal conditions (37 degrees C) during in vitro transcription, opening a route towards the construction of sensors, programmable packaging and cargo delivery systems for biomedical applications. Self-Assembling RNA Nanorings Based on RNA I/II Inverse Kissing Complexes with Associated Diceable siRNAs We experimentally characterized by biochemical and biophysical methods the formation of thermostable and ribonuclease resistant RNA nanorings which were originally designed by us using computational methods. High yields of fully programmable nanorings were produced based on several RNAI/II kissing complex variants selected for their ability to promote polygon self-assembly. This self-assembly strategy relying on the particular geometry of bended kissing complexes has potential for developing multivalent interfering RNA delivery agents. This was verified by assembling the nanoring with 6 siRNAs. These constructs were then shown to be processed by Dicer, an enzyme that is part of the RNAi silencing pathway. Specification of Protocols for the Design and Self-Assembly of siRNA Functionalized RNA Particles for Use in Automated Nanomedicine We specified three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes which are fully automatable. These NPs were engineered based on two nano-scaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs were functionalized by extension of up to 6 scaffold strands with siRNA duplexes. The assembly protocols yielded functionalized RNA NPs that we showed interacted in vitro with human recombinant Dicer to produce siRNAs. Our design strategies showed that we can provide fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs suitable for preclinical development. Using RNA Structural Flexibility Data in Nanostructure Modeling In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Our goal is to develop computer methods for aiding in this process. Our RNAJunction data base contains thousands of RNA junctions that can be used as building blocks to construct RNA nanoparticles. Two programs we developed, NanoTiler and RNA2D3D, can combine such building blocks with idealized fragments of A-form helices to produce desired 3D nanostructures. Initially, the building blocks were treated as rigid objects. Experimental data, however, shows that RNA accommodates its shape to the constraints of larger structural contexts. We included the flexibility of our building blocks into the full design process. By using an experimentally proven system, the RNA tectosquare, we showed that considering the flexibility of its kissing loop motifs as well as distortions in its helical regions appears to be necessary to achieve a realistic design. Understanding the Effects of Carbocyclic Sugars Constrained to North and South Conformations on RNA Nanodesign Relatively new types of modified nucleotides, namely carbocyclic sugars that are constrained to north or south conformations, can be used for RNA nanoparticle design to control their structures and stability by rigidifying nucleotides and altering the helical properties of RNA duplexes. Two RNA structures, an RNA dodecamer and an HIV kissing loop complex where several nucleotides were replaced with north or south constrained sugars, were studied by molecular dynamics (MD) simulations. The substituted south constrained nucleotides in the dodecamer widened the major groove and narrowed and deepened the minor groove thus inducing local conformational changes that resemble a B-form DNA helix. In the HIV kissing loop complex, north and south constrained nucleotides were substituted into flanking bases and stems. The modified HIV kissing loop complex showed a lower RMSD value than the normal kissing loop complex. The overall twist angle was also changed and its standard deviation was reduced. In addition, the modified RNA dodecamer and HIV kissing loop complex were characterized by principal component analysis (PCA) and steered molecular dynamics (SMD). PCA results showed that the constrained sugars stabilized the overall motions. The results of the SMD simulations indicated that as the backbone delta angles were increased by elongation, more force was applied to the modified RNA due to the constrained sugar analogues. Coarse Graining of RNA Nanostructures for Molecular Dynamics Simulations The modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. At best, all atom molecular dynamics studies of such molecules can obtain trajectories of a few nanoseconds duration, a limited time scale for a comprehensive characterization of such structures. A series of coarse-grained models have been developed for study of the molecular dynamics of RNA nanostructures. The models in the series have one to three beads per nucleotide and include different amounts of detailed structural information. Such a treatment allows us to reach, for systems of thousands of nucleotides, a time scale of microseconds and thus enable simulations of large RNA polymers in the context of bionanotechnology. We found that the three-beads-per-nucleotide models, described by a set of just a few universal parameters, were able to describe different RNA conformations and were comparable in structural precision to the models where detailed values of the backbone P-C4' dihedrals taken from a reference structure were included. First International Meeting on RNA Nanotechnology A meeting was held in which I was a co-organizer highlighting the recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, in Cleveland, OH. The conference was the first of its kind to bring together invited speakers in RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics.

将生物材料组织成多用途的三维组件可用于构建用于纳米医学的多功能治疗支架。我们报道了一种计算设计和实验验证三维立方纳米级支架形成的策略，该支架可以从RNA（也包括DNA和RNA/DNA杂交体）自组装，并精确控制其形状，大小和组成。这些立方纳米支架直径仅为13纳米，由短寡核苷酸组成，使其易于化学合成，点修饰和进一步功能化。通过凝胶分析、动态光散射和低温电子显微镜验证了纳米立方体的组装。通过使用荧光RNA适体，也证明了功能性RNA纳米立方体的形成，该适体仅在完全RNA组装时才具有最佳活性。我们发现，RNA纳米支架在体外转录过程中可以在等温条件下（37摄氏度）自组装，为生物医学应用的传感器、可编程包装和货物输送系统的构建开辟了一条道路。基于RNA I/II逆吻配合物与相关可切割sirna的自组装RNA纳米环我们通过生物化学和生物物理方法实验表征了我们最初使用计算方法设计的耐热性和核糖核酸酶抗性RNA纳米环的形成。基于几种RNAI/II亲和复合物变体，基于其促进多边形自组装的能力，生产了高产量的完全可编程纳米结构。这种依赖于弯曲接吻复合物的特殊几何形状的自组装策略具有开发多价干扰RNA递送剂的潜力。通过将纳米环与6个sirna组装在一起验证了这一点。这些结构随后被Dicer酶处理，Dicer酶是RNAi沉默途径的一部分。用于自动化纳米医学的siRNA功能化RNA颗粒的设计和自组装协议规范我们指定了三种组装协议，使用完全自动化的过程来生产两种不同类型的RNA自组装功能NPs。这些NPs是基于两种纳米支架设计（纳米环和纳米立方）设计的，它们作为多种sirna的载体。NPs通过延伸多达6个带有siRNA双链的支架链而实现功能化。组装方案产生了功能化的RNA NPs，我们证明了它们在体外与人重组Dicer相互作用以产生sirna。我们的设计策略表明，我们可以提供适合临床前开发的无内毒素治疗性RNA NPs的快速，经济和易于控制的生产。RNA结构柔性数据在纳米结构建模中的应用基于RNA的纳米技术这一新兴领域对结构设计过程的自动化有着迫切的需求。我们的目标是开发辅助这一过程的计算机方法。我们的RNAJunction数据库包含数千个RNA连接，这些连接可以用作构建RNA纳米颗粒的基石。我们开发的两个程序NanoTiler和RNA2D3D可以将这些构建块与理想的a型螺旋片段结合起来，产生理想的3D纳米结构。最初，构建模块被视为刚性对象。然而，实验数据表明，RNA可以根据更大结构背景的限制来调整其形状。我们将构建模块的灵活性纳入了整个设计过程。通过使用一个实验证明的系统，RNA tectosquare，我们表明，考虑到其接吻环基的灵活性以及其螺旋区域的扭曲似乎是实现现实设计所必需的。相对较新的修饰核苷酸类型，即限制为北或南构象的碳环糖，可用于RNA纳米颗粒设计，通过刚性核苷酸和改变RNA双链的螺旋性质来控制其结构和稳定性。通过分子动力学（MD）模拟研究了两种RNA结构，RNA十二聚体和HIV接吻环复合物，其中几个核苷酸被北或南约束糖取代。十二聚体中取代的南约束核苷酸使主槽变宽，使次槽变窄和加深，从而引起类似于b型DNA螺旋的局部构象变化。在HIV接吻环复合体中，北方和南方受限的核苷酸被取代到两侧的碱基和茎中。修饰后的HIV接吻环复合物的RMSD值低于正常接吻环复合物。改变了整体扭转角度，减小了其标准差。此外，利用主成分分析（PCA）和定向分子动力学（SMD）对修饰后的RNA十二聚体和HIV接吻环复合物进行了表征。主成分分析结果表明，约束糖稳定了整体运动。SMD模拟结果表明，随着主链δ角的增加，由于糖类似物的限制，修饰的RNA受到更大的力。考虑到RNA纳米结构的大小，基于RNA的纳米结构的建模和表征是一项艰巨的任务。在最好的情况下，对这类分子的所有原子分子动力学研究都可以获得持续几纳秒的轨迹，这是对这类结构进行全面表征的有限时间尺度。为了研究RNA纳米结构的分子动力学，建立了一系列的粗粒度模型。该系列中的模型每个核苷酸有一到三个小珠，并包含不同数量的详细结构信息。这样的处理使我们能够在微秒的时间尺度上达到数千个核苷酸的系统，从而可以在生物纳米技术的背景下模拟大型RNA聚合物。我们发现，由一组仅几个通用参数描述的每个核苷酸三个头的模型能够描述不同的RNA构象，并且在结构精度上与从参考结构中获取的骨干P-C4'二面体的详细值的模型相当。在俄亥俄州克利夫兰举行的第一届RNA纳米技术和治疗学国际会议上，我作为共同组织者召开了一次会议，重点介绍了RNA纳米技术的最新进展。这次会议是第一次邀请来自法国、瑞典、韩国、中国和美国各地的RNA纳米技术特邀演讲者一起讨论RNA纳米技术及其应用。它为来自学术界、政府和制药行业的研究人员提供了一个分享该领域现有知识、愿景、技术和挑战的平台，并促进了对推进这一新兴科学学科感兴趣的研究人员之间的合作。会议涵盖了一系列主题，包括生物物理和RNA纳米结构表征的单分子方法；利用化学或生化方法研究RNA纳米颗粒的结构，计算、预测和建模RNA纳米颗粒结构；RNA纳米颗粒的组装方法；用于RNA合成、偶联和标记的化学；以及RNA纳米颗粒在治疗学中的应用。