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逆接吻复合物的自组装RNA纳米纳米构造具有可溶剂的sirnaswe实验表征，其特征是生化和生物物理方法的特征，其最初是使用计算方法设计的，它们最初是由我们设计的。基于几种促进多边形自组装的能力，基于几种RNAi/II接吻复合物的变体产生了高度可编程的纳米素。这种依靠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。通过用6个siRNA组装纳米，可以验证这一点。然后证明这些构建体是由DICER处理的，DICER是RNAi沉默途径的一部分。规范siRNA官能化的RNA颗粒的设计和自组装的规程用于自动化纳米型icineicinewe，指定了三种不同类型的RNA自动化nps nps nps的纳米型icinemedicinewe指定，这些协议是完全自动化的。这些NP是根据我们的两种纳米型工具设计（Nanoring和Nanocube）设计的，它们是多个siRNA的载体。通过延伸多达6个带有siRNA双链体的脚手架链的NP通过伸展而功能化。组装方案产生了功能化的RNA NP，我们表现出与人类重组迪切尔在体外相互作用以产生siRNA的相互作用。我们的设计策略表明，我们可以提供适用于临床前开发的无内毒素治疗RNA NP的快速，经济和易于控制的产生。在建模基于RNA的纳米技术的新兴领域的纳米结构中使用RNA结构灵活性数据，需要对结构设计过程的自动化。我们的目标是开发用于协助此过程的计算机方法。我们的RNA结数据库包含数千个RNA连接，可以用作构建RNA纳米颗粒的构件。我们开发的两个程序，分别是Nanotiler和RNA2D3D，可以将这些构件与A-Corm螺旋的理想化片段相结合，以产生所需的3D纳米结构。最初，构建块被视为刚性对象。但是，实验数据表明，RNA适应了较大结构环境的约束。我们将构建块的灵活性包括在整个设计过程中。通过使用实验验证的系统，RNA TectoSquare，我们表明，考虑到其螺旋区域中其接吻循环图案的灵活性以及在其螺旋区域的扭曲似乎是实现现实设计的必要这些链的序列。这需要预测正确的链内和链间相互作用。我们开发了一个程序Nanoflower，该程序完成了这项任务。它可以包括伪诺的预测，这种情况可以解释为股间相互作用。我们表明，当应用于具有非巢碱基对的RNA复合物时，该算法的性能比其他几种结构预测方法更好。我们还使用该算法来确认预测的4链RNA纳米颗粒的自组装。无法理解碳环糖糖的影响北部和南方的构型对RNA纳米固定的构型的构型的构型，对RNA纳米固定呈现的新类型的新类型的改性型核苷酸构成了北方或北方的核心糖，是北方的含量，即北方的指定构成，构成了北方的指定，构成了构成的指定，构成了构成的构成，成分成符号，成群构成了构成的构成，成群构成了构成的构成，成群构成了构成的构成，成群构成了构成的构成，成分成分，成分成分，成分成分，成分成符号。结构和稳定性通过固化核苷酸并改变RNA双链体的螺旋特性。通过分子动力学（MD）模拟研究了两个RNA结构，一个RNA十二焦和一个HIV接吻环复合物，其中几种核苷酸被北部或南约束糖代替。十二焦点中取代的南约束核苷酸扩大了主要的凹槽，并缩小并加深了小凹槽，从而诱导了类似于B形DNA螺旋的局部构象变化。在HIV接吻环复合体中，将南北约束的核苷酸取代为侧面和茎。修改后的HIV接吻环复合物的RMSD值低于正常的接吻循环复合物。总体扭曲角度也改变了，其标准偏差减小。此外，通过主成分分析（PCA）和转导的分子动力学（SMD），修饰的RNA DodeCamer和HIV接吻环复合物的特征是。 PCA结果表明，受约束的糖稳定了总体运动。 SMD仿真的结果表明，由于伸长率增加了骨干三角角，由于糖类似物的约束，将更大的力应用于修饰的RNA上。双螺旋DNA和RNA的多刺激建模：通过谎言组的统一组统一组的建模和基于RNA的纳米结构的表征，这是一个难以实现此类结构的困难任务。从实用的角度来看，此类分子的所有原子分子动力学研究都可以获得几个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。已经开发了粗粒模型来研究RNA和DNA结构的动力学。这些模型包括不同的信息和类型。这种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Bionanotechnology的背景下启用大型RNA或DNA聚合物的模拟。在这项研究中，一种依赖于谎言组的方法用于以坐标的自由方式或必要时描述运动，以简化方程式产生的方式引入坐标。这里考虑的是双链RNA和DNA螺旋。进行了多级建模。在最粗糙的蠕虫状链中，考虑了各向异性弯曲刚度。然后表明，Bi-Rod模型以足够长的长丝长度收敛到此。在较优质水平的情况下，考虑了弹性网络，并显示了它们与更粗的水平的关系。最后，人们展示了所有原子分子动力学（细晶粒）和AFM实验结果（粗粒）如何与这些模型有关。第一次国际RNA Nanotechnologya会议举行，在该会议上，我是一项共同组织者，强调了RNA纳米技术的最新进展，这是在RNA Nanotechnology和The Shepapeistics的首次国际国际会议上介绍的，Oh Oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh oh。该会议是第一个将来自法国，瑞典，韩国，中国以及整个美国的RNA纳米技术发言人汇集的邀请发言人，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，以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科学学科的研究人员的合作。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；通过化学或生化方法，RNA纳米粒子结构的化学或生化方法，计算，预测和建模对RNA纳米颗粒的结构研究； RNA纳米颗粒组装的方法； RNA合成，结合和标记的化学； RNA纳米颗粒在治疗剂中的应用。