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.

计算机模拟设计的立方体 RNA 支架的体外组装将生物材料组织成多功能的三维组件可用于构建用于纳米医学的多功能治疗支架。我们报告了一种通过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 nm，由短寡核苷酸组成，使其适合化学合成、点修饰和进一步功能化。纳米立方体的组装通过凝胶测定、动态光散射和低温电子显微镜进行验证。还通过使用荧光RNA适体证明了功能性RNA纳米立方体的形成，该适体仅在完整RNA组装时才具有最佳活性。我们表明，RNA 纳米支架可以在体外转录过程中在等温条件（37 摄氏度）下自组装，为构建用于生物医学应用的传感器、可编程包装和货物输送系统开辟了道路。基于 RNA I/II 反向接吻复合物和相关 Diceable 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耐热和核糖核酸酶抗性 RNA 纳米环的形成，这些纳米环最初是由我们使用计算方法设计的。基于几个因其促进多边形自组装能力而选择的RNAI/II接吻复合物变体，产生了高产量的完全可编程纳米环。这种依赖于弯曲接吻复合物的特定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。这通过将纳米环与 6 个 siRNA 组装来验证。然后这些构建体被证明可以被 Dicer 加工，Dicer 是一种酶，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方案规范 我们指定了三种组装方案，以使用完全自动化的过程生产两种不同类型的 RNA 自组装功能性 NP。这些纳米颗粒是基于两种纳米支架设计（纳米结构和纳米立方体）设计的，它们可作为多种 siRNA 的载体。通过使用 siRNA 双链体延伸多达 6 条支架链，对 NP 进行功能化。组装方案产生了功能化的 RNA NP，我们证明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，我们可以快速、经济且易于控制地生产适合临床前开发的无内毒素治疗性 RNA NP。在纳米结构建模中使用 RNA 结构灵活性数据 在基于 RNA 的纳米技术的新兴领域，需要结构设计过程的自动化。我们的目标是开发计算机方法来协助这一过程。我们的 RNAJunction 数据库包含数千个 RNA 连接，可用作构建 RNA 纳米粒子的构建块。我们开发的两个程序 NanoTiler 和 RNA2D3D 可以将此类构建块与 A 型螺旋的理想化片段结合起来，以产生所需的 3D 纳米结构。最初，积木被视为刚性物体。然而，实验数据表明，RNA 可以使其形状适应更大的结构环境的限制。我们将构建块的灵活性纳入了整个设计过程。通过使用经过实验验证的系统 RNA tectosquare，我们表明，考虑到其接吻环图案的灵活性以及其螺旋区域的扭曲似乎对于实现现实的设计是必要的。了解受限于北构象和南构象的碳环糖对RNA纳米设计的影响相对较新类型的修饰核苷酸，即受限于北构象或南构象的碳环糖，可用于RNA纳米颗粒设计，通过刚性化核苷酸来控制其结构和稳定性并改变 RNA 双链体的螺旋特性。通过分子动力学 (MD) 模拟研究了两种 RNA 结构，即 RNA 十二聚体和 HIV 接吻环复合体，其中多个核苷酸被北或南限制糖取代。十二聚体中被取代的南约束核苷酸加宽了大沟，缩小并加深了小沟，从而诱导了类似于B型DNA螺旋的局部构象变化。在HIV接吻环复合体中，南北限制核苷酸被替换为侧翼碱基和茎。修饰的 HIV 接吻环复合物显示出比正常接吻环复合物更低的 RMSD 值。整体扭转角度也发生了变化，其标准偏差也减小了。此外，通过主成分分析（PCA）和引导分子动力学（SMD）对修饰的RNA十二聚体和HIV接吻环复合物进行了表征。 PCA 结果表明，受限糖稳定了整体运动。 SMD 模拟的结果表明，随着主链 δ 角因伸长而增加，由于糖类似物的限制，修饰后的 RNA 受到了更大的作用力。用于分子动力学模拟的 RNA 纳米结构的粗粒度考虑到基于 RNA 的纳米结构的尺寸，对其进行建模和表征是一项艰巨的任务。最好的情况是，此类分子的所有原子分子动力学研究都可以获得几纳秒持续时间的轨迹，这是对此类结构进行全面表征的有限时间尺度。一系列粗粒度模型被开发用于研究 RNA 纳米结构的分子动力学。该系列中的模型每个核苷酸有一到三个珠子，并包含不同数量的详细结构信息。对于包含数千个核苷酸的系统，这种处理使我们能够达到微秒的时间尺度，从而能够在生物纳米技术的背景下模拟大RNA聚合物。我们发现，由一组几个通用参数描述的每核苷酸三珠模型能够描述不同的 RNA 构象，并且在结构精度方面与包​​含主链 P-C4' 的详细值的模型相当。包括取自参考结构的二面角。第一届 RNA 纳米技术国际会议 在我作为共同组织者举行的一次会议上，重点介绍了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介绍的 RNA 纳米技术的最新进展。此次会议是同类会议中首次邀请来自法国、瑞典、韩国、中国和美国各地的 RNA 纳米技术领域的特邀演讲者齐聚一堂，讨论 RNA 纳米技术及其应用。它为学术界、政府和制药行业的研究人员提供了一个平台，分享该领域的现有知识、愿景、技术和挑战，并促进有兴趣推进这一新兴学科的研究人员之间的合作。会议涵盖了一系列主题，包括用于表征 RNA 纳米结构的生物物理和单分子方法；通过化学或生物化学方法对RNA纳米粒子进行结构研究，计算、预测和建模RNA纳米粒子结构； RNA纳米粒子的组装方法； RNA 合成、缀合和标记化学； RNA纳米颗粒在治疗学中的应用。