Computational RNA Nanodesign

计算RNA纳米设计

• 批准号: 7966010
• 负责人: Bruce Shapiro
• 金额: $ 64.3万
• 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/7966010
• 关键词: Affect; Algorithms; Back; Base Pairing; Behavior; Biocompatible Materials; Book Chapters; Canada; Cereals; Complex; Computing Methodologies; DNA; Databases; Dependence; Detection; Development; Dimensions; Disease; Elasticity; Elements; Environment; Freedom; Generations; Genetic; Goals; Graph; High temperature of physical object; Hydration status; Hydrogen Bonding; Ions; Length; Malignant Neoplasms; Measures; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Motion; Mutate; Mutation; Nanostructures; Ontario; Process; Property; Protein Binding; Publishing; RNA; RNA Databases; RNA Sequences; Receptor Cell; Recurrence; Research; Series; Side; Simulate; Solvents; Specific qualifier value; Structure; System; Temperature; Testing; Therapeutic; Time; Universities; Variant; Water; base; catalyst; combinatorial; design; improved; inorganic phosphate; molecular dynamics; molecular mechanics; nanobiology; nanodevice; nanoparticle; nanosecond; programs; research study; satisfaction; self assembly; software systems; synthetic biology; three dimensional structure; uptake

<b>Nanotiler</b> 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. A specified set of motifs can be placed in space and joined with A-form helix connectors. The connector lengths can be varied. This leads to a large combinatorial space of structures. Some of these structures form closed rings; others can form dendrimer-like conformations. Ring-formation can be detected automatically. Constraint satisfaction methods can be applied to improve ring closure and proper fit of connected helices. In addition, a graph that indicates the desired topology can be input into the design process of a structure. A graph matching algorithm is used to determine when a designed structure matches the desired topology. 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. A sequence-fusing algorithm connects fragments that were used in the generation of the conformational topologies. Next the sequence optimization algorithm can be 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. This is accomplished by an algorithm that searches known structures for the same base pairs that have a conformation similar to that needed in the generated 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. <P> <b>Molecular Dynamics Characterization of the Hexagonal RNA Nanoring</b> 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. <P> <b>Mesoscopic model</b> 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. <P> <b>Rational Design of RNA Nanostructures</b> 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 conne [summary truncated at 7800 characters]

为了更好地促进基于RNA的纳米颗粒的设计，开发了一个软件系统NanoTiler，允许在几个不同的概念层面上进行RNA纳米设计。NanoTiler的一个关键特征是它能够利用来自RNAJunction数据库的基序来完成3D RNA结构空间的组合搜索。一组特定的图案可以放置在空间中，并与A型螺旋连接器连接。连接器的长度可以改变。这导致了一个大的结构组合空间。其中一些结构形成闭合环；其他的可以形成树突状构象。环状结构可以自动检测。约束满足法可以改善环的闭合性和连接螺旋的适当配合。此外，还可以在结构的设计过程中输入指示所需拓扑的图形。图匹配算法用于确定设计的结构何时与期望的拓扑匹配。一旦实现了理想的拓扑结构，NanoTiler就可以专注于产生一组序列，这些序列可以通过实验测试来形成所设计的结构。序列融合算法将用于生成构象拓扑的片段连接起来。其次，可以应用序列优化算法来限制设计序列之间的串扰量。理想情况下，每个单独设计的序列应该以无障碍的方式折叠成其所需的构象（序列片段的目标二级结构表示）。序列是重复突变的，除了那些必须保持重要基序的部分，如从我们的RNAJunction数据库中获得的那些，对每组突变序列进行评分。NanoTiler与其他程序一起测量序列之间发生的杂交程度和折叠成目标二级结构的程度。一旦生成了一组优化的序列，突变就会被替换回3D结构中。这是通过一种算法来完成的，该算法在已知结构中搜索具有与生成结构中所需构象相似的相同碱基对。一旦所有的片段都设计好了，它们就会受到分子力学的影响，以固定键的长度和角度。最后，如果需要，整个结构或部分结构将受到分子动力学的影响，以表征所设计的纳米结构的动态特性。六角形RNA纳米结构的分子动力学表征在过去几年中，在理解RNA结构方面取得了重大进展，导致了对任意大小RNA纳米结构的自组装的RNA结构学的研究。我的团队设计了一个RNA六边形环和纳米管，由六个a形螺旋边和六个接吻环基序组成，每个角的角度接近120度，从而可以形成六边形。这个环的形成已被实验证明。一个问题是纳米环的计算表征。与加拿大安大略省威尔弗里德劳里埃大学的罗德里克梅尔尼克一起，我们对六边形环进行了全原子分子动力学研究。由于它的大小，这种计算需要很长时间，并且必须是有限的持续时间。我们想确定纳米环的稳定性如何受到温度、反离子和溶剂的影响，以及纳米环如何受到外力的影响。结果表明，环在310°K时似乎是稳定的，而在510°K时，正如预期的那样，环似乎在向展开的单个碎片的方向坍缩成致密的球状状态。在较高的温度下，环的水化作用减少。在510 k时，环中氢键的相互作用也有明显的损失，磷酸盐在更高的温度下变得更紧密。我们还发现了一个令人惊讶的现象，即随着温度的升高，离子的吸收也在增加。这可能是由于水的介电常数对温度的依赖。我们发现纳米环对二维平面内压缩的拉伸弹性估计低于典型的软物质代表，如DNA。< < & b&gt；介观模型&lt；/b&gt；考虑到基于rna的纳米结构的尺寸，建模和表征是一项艰巨的任务。我的团队之前设计的RNA六边形纳米环和纳米管就是一个例子。在最好的情况下，对这类分子的所有原子分子动力学研究都可以获得持续几纳秒的轨迹，这是对这类结构进行全面表征的有限时间尺度。我们与加拿大安大略省威尔弗雷德劳里埃大学的罗德里克·梅尔尼克的团队合作，一直在开发RNA的粗粒度模型，这种模型可以更容易地表征RNA纳米颗粒中常见的大结构。利用分子动力学开发并模拟了一系列基于3个珠子（每个珠子代表一组有助于系统结构行为的原子）的模型。目标是获得通用参数，这些参数可以表示像捕获珠子之间存在的相互作用的粗粒度实体这样的大型结构。这种粗粒度处理使我们能够获得比全原子分子动力学模拟长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。这种头模型的参数化需要从实验和完全原子分子动力学中获得的信息。增加复杂性的是，除了常见的沃森-克里克碱基对之外，RNA结构还包含各种各样的相互作用。纳米环举例说明了这一问题，因为它不仅包含沿其边缘的a型沃森-克里克相互作用，而且在由接吻环接触组成的角上也有非规范的非a型相互作用。我们的结果表明，3头模型的变体表现得相当好，并且只需要包含有关磷酸盐- c4二面体自由度的细节才能得到良好的表示。RNA纳米结构的合理设计&lt；/b&gt；在最近受邀出版的两本书章节中，我们列出了合理设计RNA纳米结构的基本原则。了解天然RNA分子如何折叠和组装是一个基本要素。据推测，一些RNA序列具有在其自然环境之外自主折叠成精确的三维结构的能力。这些折叠被称为基序，经常在RNA结构数据库中找到。然而，并非所有解出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在其自然环境中折叠和组装。因此，在设计基于rna的纳米结构时必须考虑几个问题。这些问题包括：1)给定结构是否为主题？2)母题是否在多个结构中反复出现？3)母题是否能够在其自然语境之外形成？4)母题在自然语境之外的稳定性如何？5)当与其他母题或螺旋锥相关联时，母题的稳定性如何[摘要截短为7800字]