Computational RNA Nanodesign

计算RNA纳米设计

• 批准号: 8157607
• 负责人: Bruce Shapiro
• 金额: $ 74.37万
• 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/8157607
• 关键词: RNA

Nanotiler To better facilitate the design of RNA based nanoparticles a software system, NanoTiler, was developed that permits RNA nanodesign at several different conceptual levels. A key feature of NanoTiler is its ability to accomplish combinatorial search of 3D RNA structure spaces by utilizing motifs derived from our RNAJunction database or designed de novo. Once a desired topology is realized, NanoTiler can then focus on producing a set of sequences that can be experimentally tested for the formation of the designed structure. The sequence optimization algorithm is applied to limit the amount of cross talk between the designed sequences. Ideally, each individually designed sequence should fold in an unobstructed fashion into its desired conformation (a target secondary structure representation of the sequence fragment. Sequences are repeatedly mutated, except for the portions that have to be maintained to preserve important motifs such as those obtained from our RNAJunction database, scoring each set of mutated sequences. NanoTiler in conjunction with other programs measures the degree of hybridization that occurs between the sequences and the degree of folding into the target secondary structure. Once an optimized set of sequences is generated, mutations are substituted back into the 3D structure. Once all fragments are designed, they are subjected to molecular mechanics to fix bond lengths and angles. Finally, if desired, the entire structure or portions of the structure are subjected to molecular dynamics to characterize the dynamical qualities of the designed nanostructure. Using the above described methodology RNA and RNA/DNA hybrid nanocubes were designed and experimentally proven to self-assemble. These cubes were composed of 6 short or 10 short oligonucleotides that make them amenable to chemical synthesis, point modifications and further functionalization. The nanocube assemblies were verified by gel assays, dynamic light scattering and cryogenic electron microscopy. In addition, the cubes were functionalized by the incorporation of fluorescent aptamers that would only light up upon proper cube assembly. It was also shown that the cubes can assemble under isothermal conditions at 37 degree centigrade during in vitro transcription which opens a route towards the construction of sensors, programmable packaging and cargo delivery systems. Molecular Dynamics Characterization of the Hexagonal RNA Nanoring The significant progress made over the past several years in understanding RNA structure, has led to research into RNA architectonics that deals with the self-assembly of RNA nanostructures of arbitrary size. My group designed an RNA hexagonal ring and nanotube composed of six A-form helical sides and six kissing loop motifs that approximate a 120 degree angle at each corner thus allowing the formation of hexagons. Formation of this ring has been demonstrated experimentally. An issue is the computational characterization of the nanoring. In conjunction with Roderick Melnik from the Wilfrid Laurier University in Ontario, Canada we have been performing all-atom molecular dynamics studies of the hexagonal ring. Because of its size such calculations take a long time and must be of limited duration. We wanted to determine how the stability of the nanoring was affected by temperature, counter-ions and solvent and how the nanoring is affected by external forces. Results indicate that the ring appears to be stable at 310 degrees K, while at 510 degrees K, as might be expected, the ring seems to be collapsing into a compact globular state on its way to unfolded single fragments. There was reduced hydration of the ring at the higher temperature. There was also a significant loss of hydrogen bond interactions in the ring at 510 degrees K. Phosphates became closer together at the higher temperature. We also found a surprising phenomenon where there was an uptake of ions as a function of increasing temperature. This may be due to the dependence of the water dielectric constant on temperature. We found that an estimate of the tensile elasticity of the nanoring against 2D in-plane compression was lower than a typical soft matter representative such as DNA. Mesoscopic model The modeling and characterization of RNA-based nanostructures is a difficult task given the size of such structures. This is exemplified by my groups previously designed RNA hexagonal nanoring and nanotube. 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. In conjunction with Roderick Melnik's group at the Wilfred Laurier University in Ontario, Canada we have been developing coarse-grained models of RNA that can be used to more easily characterize the large structures that are often found in RNA nanoparticles. A series of models based on 3 beads (each bead represents a group of atoms that contribute to the structural behavior of the system) were developed and simulated using molecular dynamics. The goal is to obtain universal parameters that can represent such large structures as coarse-grained entities that capture the interactions that exist between the beads. Such a coarse-grained treatment has allowed us to obtain microsecond time scales that are three orders of magnitude longer than all-atom molecular dynamics simulations. This methodology is allowing us to study the slowest conformational motions of the RNA. Parameterization of such bead models requires information obtained from experiments as well as from full atomistic molecular dynamics. What adds complexity is that RNA structures contain a wide variety of interactions beyond the commonly found Watson-Crick basing pairs. The nanoring exemplifies this issue because it not only contains A-form Watson-Crick interactions along its edges but also has non-canonical non-A-form interactions in the corners that are composed of kissing loop contacts. Our results indicate that variants of the 3-bead model perform reasonably well and that the inclusion of only the details about the Phosphate-C4 dihedral degrees of freedom is needed for a good representation. Rational Design of RNA Nanostructures In two recently invited published book chapters we laid out the basic principles for the rational design of RNA nanostructures. An understanding of how natural RNA molecules fold and assemble is an essential element. It is assumed that some RNA sequences have the ability to fold autonomously into precise 3-D structures outside of their natural context. These folds are called motifs and are often found in the database of RNA structures. However, not all solved structures are autonomous folding domains. Therefore it is still difficult to determine whether given sequences will fold and assemble as expected out of their natural context. Thus, several questions must be considered when designing RNA-based nanostructures. These include: 1) Is a given structure a motif? 2) Is the motif recurrent within multiple structures? 3) Is the motif able to form outside its natural context? 4) What is the stability of the motif outside its natural context? 5) what is the stability of the motif when associated with other motifs or helical connectors? 6) What is the relative flexibility of a motif within the desired framework? 7) How do environmental factors such as temperature and ion concentration affect stability? Approaches are discussed in the two chapters which attempt to answer some of these questions and layout an integrated approach of experimental and computational RNA nanodesign.

为了更好地促进基于RNA的纳米颗粒的设计，纳米动物的纳米粒子纳米构成了纳米动物，该软件系统允许在几个不同的概念水平上进行RNA纳米设计。纳米动物的一个关键特征是它通过利用从我们的RNAJunction数据库或从头设计的DE DE DE DE NOVE的图案来完成对3D RNA结构空间的组合搜索的能力。 一旦实现了所需的拓扑结构，纳米动物就可以专注于产生一组序列，这些序列可以通过实验测试，以形成设计的结构。应用序列优化算法以限制设计序列之间的交叉谈话量。理想情况下，每个单独设计的序列都应以毫无疑问的方式折叠成所需的构象（序列片段的目标二级结构表示。序列反复突变，除了必须维护以保留重要主题的部分，例如从我们的RNAJUNTICT数据库，对每个突变序列进行评分。回到3D结构。设计的纳米结构。这些立方体由6个短或10个短寡核苷酸组成，使其与化学合成，点修饰和进一步的功能化。通过凝胶测定，动态光散射和低温电子显微镜验证纳米管组件。此外，通过掺入荧光体能力，只能在适当的立方体组件上亮起荧光体能力。还表明，在体外转录期间，在等温条件下，这些立方体可以在等温条件下组装，这为传感器，可编程包装和货物输送系统的构建提供了途径。六边形RNA的分子动力学表征在过去几年中，在理解RNA结构方面取得的重大进展已导致对RNA架构的研究，这些RNA架构涉及涉及任意大小的RNA纳米结构的自组装。我的小组设计了一个RNA六角形环和纳米管，该环由六个A形螺旋侧和六个接吻环基序组成，每个角落在每个角处近似于120度角，从而允许形成六边形。该环的形成已通过实验证明。一个问题是Nanoring的计算表征。与加拿大安大略省Wilfrid Laurier大学的Roderick Melnik一起，我们一直在进行六角形环的全原子分子动力学研究。由于其大小，这种计算需要很长时间，并且持续时间必须有限。我们想确定Nanoring的稳定性如何受到温度，反离子和溶剂的影响，以及纳米对外力的影响。结果表明，该环在310度K下似乎是稳定的，而在510度K处，该环在可能的情况下似乎在展开的单个片段的途中塌陷成紧凑的球状状态。在较高温度下，环的水合减少。在510摄氏度的环中，在较高温度下，在510摄氏度的环中也有明显的氢键相互作用。我们还发现了一个令人惊讶的现象，其中有离子的摄取是温度升高的函数。这可能是由于水介电常数对温度的依赖性所致。我们发现，对2D平面压缩的Nanoring的拉伸弹性估计低于典型的软物质代表，例如DNA。鉴于此类结构的大小，介观模型基于RNA的纳米结构的建模和表征是一项艰巨的任务。我的组以前设计的RNA六角形纳米型和纳米管为此说明了这一点。充其量，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。与加拿大安大略省Wilfred Laurier大学的Roderick Melnik的小组一起，我们一直在开发RNA的粗粒细粒度模型，这些模型可用于更容易地表征RNA纳米颗粒中经常发现的大型结构。使用分子动力学开发并模拟了一系列基于3个珠子（每个珠子代表有助于系统结构行为的原子）。目的是获得可以代表大型结构的通用参数，例如捕获珠子之间存在相互作用的粗粒实体。这种粗粒的处理使我们获得了比全原子分子动力学模拟的三个数量级的微秒时间尺度。这种方法使我们能够研究RNA最慢的构象运动。此类珠模型的参数化需要从实验以及完全原子分子动力学中获得的信息。补充的复杂性是，RNA结构包含除了常见的Watson-Crick Basing对之外的各种相互作用。 Nanoring例证了这个问题，因为它不仅包含A-Form Watson-Crick沿其边缘的相互作用，而且还包含由亲吻环触点组成的角落中的非典型的非A形式相互作用。我们的结果表明，3珠模型的变体表现出色，并且仅包含有关磷酸盐C4二面体自由度的细节才能进行良好的表示。 RNA纳米结构的合理设计在最近被邀请的两本书章节中，我们列出了RNA纳米结构合理设计的基本原理。了解天然RNA分子如何折叠和组装是必不可少的元素。假定某些RNA序列具有在自然背景之外自主折叠成精确的3-D结构的能力。这些褶皱称为基序，通常在RNA结构数据库中找到。但是，并非所有已解决的结构都是自主折叠域。因此，仍然很难确定给定的序列是否会按照自然背景中的预期折叠和组装。因此，在设计基于RNA的纳米结构时必须考虑几个问题。其中包括：1）给定的结构是主题吗？ 2）主题是否在多个结构中复发？ 3）主题能够在自然背景之外形成吗？ 4）在自然背景之外，主题的稳定性是什么？ 5）当与其他主题或螺旋连接器相关联时，主题的稳定性是什么？ 6）在所需框架内，主题的相对灵活性是什么？ 7）温度和离子浓度等​​环境因素如何影响稳定性？在两章中讨论了方法，这些章节试图回答其中一些问题并布局实验和计算RNA纳米设计的综合方法。