Computational and Experimental RNA Nanobiology

计算和实验 RNA 纳米生物学

• 批准号: 8552960
• 负责人: Bruce Shapiro
• 金额: $ 83.86万
• 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/8552960
• 关键词: Academia; Algorithms; Automation; B-DNA; Base Pairing; Biochemical; Biocompatible Materials; Cereals; Chemicals; Chemistry; China; Collaborations; Complex; Computing Methodologies; DNA; DNA Structure; Data; Databases; Detection; Development; Dicer Enzyme; Dimensions; Discipline; Disease; Double-Stranded RNA; Drug Industry; Endotoxins; Engineering; Environment; Equation; Experimental Designs; Filament; France; Genetic; Goals; Government; HIV; Human; In Vitro; International; Knowledge; Label; Length; Major Groove; Malignant Neoplasms; Methodology; Methods; Minor Groove; Modeling; Molecular; Molecular Conformation; Motion; Nanostructures; Nanotechnology; Normal Range; Nucleotides; Pathway interactions; Polymers; Principal Component Analysis; Process; Production; Property; Protein Binding; Protocols documentation; RNA; RNA I; RNA Interference; RNA chemical synthesis; Receptor Cell; Recombinants; Research; Research Personnel; Resistance; Ribonucleases; Shapes; Small Interfering RNA; South Korea; Specific qualifier value; Structure; Sweden; System; Technology; Testing; Therapeutic; Time; United States; Variant; Vertebral column; Vision; analog; base; catalyst; design; flexibility; human DICER1 protein; interest; meetings; molecular dynamics; multi-scale modeling; multilevel analysis; nano; nanobiology; nanobiotechnology; nanodevice; nanoparticle; nanosecond; particle; pre-clinical; programs; retinal rods; scaffold; self assembly; simulation; single molecule; stem; sugar; symposium; synthetic biology

Self-Assembling RNA Nanorings Based on RNA I/II Inverse Kissing Complexes with Associated Diceable siRNAsWe 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 NanomedicineWe 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 of our 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 ModelingIn 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.Multistrand RNA Structure Prediction and Nanostructure Design including PseudoknotsOne of the steps required for determining the proper set of RNA strands that will self-assemble into a desired RNA nanostructure is to determine the sequences of these strands. This requires the prediction of the correct intra-strand and inter-strand interactions. We developed a program, NanoFolder, which accomplishes this task. It can include the prediction of pseudoknots, which is this case can be interpreted as inter-strand interactions. We showed that this algorithm, performs better than several other structure prediction methods when applied to RNA complexes with non-nested base-pairs. We also experimentally confirmed the self-assembly of a predictied 4-stranded RNA nanoparticle using this algorithm.Understanding the Effects of Carbocyclic Sugars Constrained to North and South Conformations on RNA NanodesignRelatively 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.Multiscale Modeling of Double-Helical DNA and RNA: A Unification through Lie GroupsThe modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. From a practical stand point, all atom molecular dynamics studies of such molecules can obtain trajectories of several nanosecond durations, a limited time scale for a comprehensive characterization of such structures. Coarse-grained models have been developed to study the dynamics of RNA and also DNA structures. The models include different amounts and types of information. Such a treatment will ultimately allow us to study systems consisting of thousands of nucleotides, at time scales of microseconds and thus enable simulations of large RNA or DNA polymers in the context of bionanotechnology. In this research, a method that relies on Lie groups was used to describe motions in a coordinate free way or when necessary, coordinates are introduced in a way in which simplified equations result. What was considered here were double stranded RNA and DNA helices. Multilevel modeling was done. At the coarsest level worm-like chains with anisotropic bending stiffness were considered. It was then shown that bi-rod models converge to this for sufficiently long filament lengths. At yet finer levels elastic networks were considered and it was shown how they related to coarser levels. Finally it was shown how all atom molecular dynamics (fine grain) and AFM experimental results (coarse grain) relate to these models.First International Meeting on RNA NanotechnologyA 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.

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设计的耐高温和耐核糖核酸酶的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，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NPs。这些NPs是基于我们的两种纳米支架设计（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结构柔韧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。包括假结在内的多链RNA结构预测和纳米结构设计确定RNA链自组装成所需RNA纳米结构的适当集合所需的步骤之一是确定这些链的序列。这需要正确预测链内和链间的相互作用。我们开发了一个程序NanoFolder来完成这个任务。它可以包括假结的预测，在这种情况下可以解释为链间相互作用。我们证明，当应用于非嵌套碱基对的RNA复合物时，该算法比其他几种结构预测方法表现更好。我们还通过实验证实了使用该算法预测的4链RNA纳米颗粒的自组装。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。双螺旋DNA和RNA的多尺度建模：通过李群的统一基于RNA的纳米结构的建模和表征是一项艰巨的任务，因为这种结构的大小。从实际的角度来看，这些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，这是对这些结构进行全面表征的有限时间尺度。粗粒度模型已被用于研究RNA和DNA结构的动力学。这些模型包含了不同数量和类型的信息。这样的处理最终将使我们能够在微秒的时间尺度上研究由数千个核苷酸组成的系统，从而在生物纳米技术的背景下模拟大型RNA或DNA聚合物。在本研究中，我们使用了一种依赖李群的方法来以无坐标的方式描述运动，或者在必要时，以简化方程的方式引入坐标。这里考虑的是双链RNA和DNA螺旋。进行了多层次建模。在最粗的层次上考虑具有各向异性弯曲刚度的蠕虫状链。然后证明了双棒模型在足够长的灯丝长度下收敛于此。在更精细的层次上，我们考虑了弹性网络，并展示了它们是如何与更粗糙的层次相关联的。最后说明了原子分子动力学（细粒）和原子力显微镜实验结果（粗粒）与这些模型的关系。第一届RNA纳米技术国际会议召开了一次会议，我是会议的共同组织者，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，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。