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无障碍的方式折叠成其所需的构象（目标的二级结构表示序列片段的二级结构表示。序列反复进行突变，除了必须维护重要的主题以保持重要的主题，例如从我们的rnajunjuntion数据库中获得的每个程序量表的序列序列，以确定了conjunter sections seles nannanoters seles nannanoter secters nanjuntized secters secters。序列和目标二级结构的折叠程度。 RNA/DNA杂交纳米烟是由6个短或10个短或10个短的寡核苷酸组成的，这些寡核苷酸可与化学合成，纳米型组装验证，并通过凝胶分析和冷冻量进行综合。只有在适当的立方体组件上点亮的适体也表明，在体外转录期间，在37度的等温条件下，可以在等温条件下组装，这为传感器的构建，可编程包装和货物递送系统的构建提供了多大的发展。我组的RNA纳米结构由六个A形的螺旋侧和六个接吻循环图案组成，在每个角落都有六个角度的形成。加拿大安大略省的Wilfrid Laurier大学对六角形戒指进行了全部原子分子动力学研究，因为它的尺寸需要很长时间，并且必须是有限的。可以预期的是，在较高的温度下，环在较高的温度下，磷酸盐在510度时，环在较高的温度下，环上的氢相互作用也很明显。水介电常数对温度的依赖性，对2D的平面压缩的拉伸弹性比典型的软物质代表（例如DNA）的模型，而基于RNA的纳米结构的表征是由我的纳米结构进行的最好的，所有对这种分子的分子动力学研究可以获得几个纳秒持续时间的轨迹，这是有限的时间尺度，用于对此类结构的全面表征基于3个珠子的模型（每个珠子代表了有助于系统结构行为的一组原子），并使用分子动力学进行了模拟。这种方法使我们能够研究此类珠模型的最缓慢的构象运动。 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纳米结构对自然的RNA分子的理解是一个必不可少的元素。因此，在设计基于RNA的纳米结构时必须考虑几个问题。 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。