Computational and Experimental RNA Nanobiology

用于RNA纳米技术应用的工程RNA纳米设计：特邀评论（论文和章节）比较和对比了RNA和DNA纳米技术之间的一些差异，强调了使用RNA作为开发基于RNA的纳米粒子的一种方式所获得的优势。在了解RNA的生物结构和功能方面的多年经验使基于RNA的功能纳米颗粒的设计成为可能，并使这些颗粒达到临床前水平。大量来自自然界和新设计的RNA基序可用于自组装各种RNA形状。功能可以从sirna，核开关，核酶和RNA适体中收集，仅举几例。讨论了两种主要的设计方法，RNA架构和单链瓦片方法。RNA结构依赖于RNA构建块的二级结构形成，而单链瓦片方法试图将RNA二级结构减少到最低限度，但依赖于相对较短的链间螺旋相互作用。总结了RNA纳米设计的计算方法，包括我们的软件，NanoTiler， RNA2D3D和RNAjunction数据库。我们对RNA纳米颗粒的共转录组装方法进行了总结，这些纳米颗粒可能含有核酸酶保护的修饰碱基（见下文）。讨论了针对各种基因的sirna的RNA纳米结构和纳米立方的功能化，并总结了一种涉及使用具有分裂功能的RNA/DNA杂交体的新方法（见下文）。在纳米结构建模中使用RNA结构柔韧性数据：在基于RNA的纳米技术这一新兴领域中，需要实现结构设计过程的自动化。最初，用于构建RNA纳米结构的构建块被视为刚性物体。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。此外，我们描述了一种使用各向异性网络建模的粗粒度方法，以显着加快确定RNA纳米结构固有的主要运动的过程。与动态光散射实验相比，该程序能够解释结构尺寸变化以及与不同细微序列变化相关的热力学稳定性。用sirna功能化的化学修饰的RNA纳米结构的共转录组装：我们开发了一种一锅共转录组装技术，该技术使用T7 RNA聚合酶在DNA模板上操作，与锰离子结合，产生功能化的RNA纳米颗粒，其中可以包括修饰的碱基，保护颗粒免受人类血清中核酸酶的影响。该方法有效地允许相对较大的RNA纳米结构的廉价组装。在生理条件下完成了具有多个sirna功能化的复杂纳米结构（一些包含多达22条链）的组装。sirna与支架相关。对核酸酶的保护作用得到了显著的提高，不同的合成颗粒在有或没有修饰碱基的情况下都表现出良好的沉默性。激活RNA-DNA杂交体的分裂功能：利用含有分裂功能的初始失活RNA-DNA杂交体，我们开发了一种方法，通过将DNA和RNA重新结合到其相关的RNA和DNA双工产物中来激活功能。重组是由互补杂交体的独立传递触发的，每个杂交体都含有互补的DNA支点。在此基础上，产生了功能性RNA复合物和功能性DNA复合物。这些产物比它们的起始杂化双相反应物在热力学上更稳定。该方法具有以下几个优点：1)条件激活吐槽功能；2)对血液中核酸酶的保护；3) DNA和RNA可以功能化；4)重缔合动力学可由支点长度控制；5)通过将分子信标附着在DNA链上实现实时跟踪；6)多种分裂功能的使用，即DNA和RNA分裂功能可以在重新结合时被激活；7)混合双工可以独立靶向。使用这种方法，我们证明了该方法在体外和体内都有效，在细胞中发生可切割sirna的重新关联，同时靶基因沉默。除乳腺癌细胞系中的eGFP外，靶基因还包括HIV-1中的蛋白酶和包膜基因，这些基因在感染细胞中显示出病毒水平和相关病毒蛋白的显著降低。此外，毒性水平很低。此外，A549肺腺癌细胞中的一种癌症基因（GSTP1）在使用适当设计的杂交体分别在不同的两天共转染后，显示出GTSP1蛋白的显著减少。杂交体的生物分布研究表明，在乳腺癌小鼠模型中，肿瘤摄取显著，eGFP沉默显著。杂交鼠对血液中的小鼠核酸酶也有抵抗力。作为原理的进一步证明，一个分裂失活的孔雀石绿适体在杂交重组后被激活。为了克服siRNA递送中的障碍，如穿越细胞膜和在血液中的半衰期短，我们研究了亲bolaamphiles与siRNA络合作为递送剂的使用。亲水分子由两个带正电的头基组成，它们位于内部疏水链的两侧。它们具有相对较低的毒性，在血液中的持久性较长，并且能够在与sirna相关的水条件下形成多阳离子胶束。研究人员比较了两种具有乙酰胆碱头基团以两种不同构型连接到烷基链上的亲水分子GLH-19和GLH-20与sirna复合物并将其传递到细胞中诱导基因沉默的能力。分子动力学模拟表明，由于静电、氢键和疏水相互作用，亲水分子与sirna结合。这些计算机研究得到了各种体外、细胞培养和体内研究的支持。MD模拟预测了与GLH-19胶束相关的sirna对核酸酶降解的更好保护。MM-PBSA和MM-GBSA方法预测GLH-19/siRNA复合物具有更高的结合亲和力。与这些计算结果一致，凝胶实验表明GLH-19/siRNA复合物的结合更强，相互作用更稳定，并且在核酸酶存在下几乎没有降解。低温电镜研究表明，GLH-19/siRNA和GLH-20/siRNA具有不同的相对疏水性，GLH-19/siRNA疏水性较弱。基于细胞培养转染和活体荧光成像结果，我们认为GLH-19和GLH-20都具有作为siRNA递送载体的巨大潜力，其中GLH-19是更好的候选载体。Bola/siRNA复合物显著提高了siRNA的化学稳定性，并提供了良好的细胞内摄取，随后是特异性基因沉默。此外，根据不同的应用，siRNA的化学保护程度可以通过简单地改变载体来改变。

Engineered RNA nanodesigns for applications in RNA nanotechnology: Invited reviews (paper and chapter) compare and contrast some of the differences between RNA and DNA nanotechnologies emphasizing the advantages to be attained from the use of RNA as a modality for developing RNA-based nanoparticles. Years of experience in understanding RNAs' biological structure and function has enabled the design of RNA based functional nanoparticles and have brought these particles to preclinical levels. A multitude of RNA motifs from nature and de novo designs can be used for self-assembling various RNA shapes. Functionalities can be gleaned from siRNAs, riboswitches, ribozymes and RNA aptamers to name a few. Two major design approaches are discussed, RNA architectonics and a single-stranded tiles approach. RNA architectonics relies on secondary structure formation of the RNA building blocks while the single-stranded tiles approach attempts to reduce RNA secondary structure to a minimum but relies on relatively short inter-strand helical interactions. Computational approaches to RNA nanodesign are summarized, including our software, NanoTiler, RNA2D3D, and the RNAjunction database. Our methods for co-transcriptional assembly of RNA nanoparticles that may contain modified bases for nuclease protection is summarized (see below). Functionalization of our RNA nanorings and nanocubes with siRNAs targeting various genes are discussed as well as a new methodology involving the use of RNA/DNA hybrids with split functionalities is summarized (also below). Using RNA structural flexibility Data in Nanostructure Modeling: In the emerging field of RNA-based nanotechnology there is a need for automation of the structure design process. Initially, the building blocks used to build our RNA nanostructures 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. In addition, we describe a coarse grained method using anisotropic network modeling to significantly speed up the process of determining the main motions that are inherent in RNA nanostructures. The procedure is able to account for structure size variations compared to dynamic light scattering experiments and the thermodynamic stabilities associated with different subtle sequence variations. Co-transcriptional assembly of chemically modified RNA nanostructures functionalized with siRNAs: We developed a one-pot co-transciptional assembly technique that uses T7 RNA polymerase operating on DNA templates in conjunction with manganese ions to produce functionalized RNA nanoparticles that can include modified bases that protect the particles from nucleases found in human blood serum. The methodology effectively permits the inexpensive assembly of relatively large RNA nanoconstructs. Assemblies done under physiological conditions of complex nanostructures (some containing as many as 22 strands) functionalized with multiple siRNAs were accomplished. The siRNAs were associated with the scaffolds. Nuclease protection was significantly improved, and the different synthesized particles were shown to silence well with and without the modified bases. Activation of split functionalities on reassociation of RNA-DNA hybrids: Using initially inactive RNA-DNA hybrid duplexes containing split functionalities, we developed a methodology that enables the activation of the functionalites by reassociation of the DNA and RNA into their associated RNA and DNA duplex products. Reassociation is triggered by the independent delivery of complementary hybrids that each contain complementary DNA toeholds. Upon reassociationfunctional RNA complexes and functional DNA complexes are produced. These products are more thermodynamically stable than their starting hybrid duplex reactants. This novel approach has several advantages 1) conditional activation of spit functionalities; 2) protection against nucleases in the blood; 3) DNA and RNA can be functionalized; 4) kinetics of reassociation can be controlled by toehold length; 5)real time tracking can be accomplished by attaching molecular beacons to the DNA strands; 6) use of multiple split functionalities i.e. DNA and RNA split functionalities can become activated upon reassociation and; 7) hybrid duplexes can be targeted independently. Using this approach we showed that the methodology works in vitro and in vivo where reassociation of diceable siRNAs occurred in cells with concomitant silencing of the targeted genes. Genes targeted, besides eGFP in a breast cancer cell line, were the protease and the envelope genes in HIV-1, which showed very significant reductions in virus levels and associated viral proteins in infected cells. In addition toxicity levels were low. Also, a cancer gene (GSTP1) in A549 lung adenocarcinoma cells showed a significant decrease in GTSP1 protein using appropriately designed hybrids individually co-tranfected on two different days. Biodistribution studies of the hybrids showed significant tumor uptake in xenograph breast cancer mouse models as well as significant silencing of eGFP. Hybrids were also resistant to mouse nucleases in the bloodstream. As a further proof of principle, a split inactive malachite green aptamer was shown to be activated upon hybrid recombination. In silico, in vivo and in vitro studies of bolaamphiphiles for potential therapeutic siRNA delivery: To overcome obstacles in siRNA delivery, such as crossing cellular membranes and short half-lives in blood, we studied the use of bolaamphiles complexed with siRNA as a delivery agent. Bolaamphiphiles consist of 2 positively charged head-groups that flank an internal hydrophobic chain. They have relatively low toxicities, long persistence in the blood and have the ability to form poly-cationic micelles under aqueous conditions that associate with siRNAs. Two different bolaamphiphiles with acetylcholine head groups attached to an alkyl chain in two distinct configurations, GLH-19 and GLH-20, were compared for their abilities to complex with siRNAs and deliver them into the cells inducing gene silencing. Molecular dynamics simulations showed that bolaamphiphiles associate with siRNAs due to electrostatic, hydrogen bonding, and hydrophobic interactions. These in silico studies were supported by various in vitro, cell culture and in vivo studies. MD simulations predicted better protection against nuclease degradation for siRNAs associated with the GLH-19 micelles. MM-PBSA and MM-GBSA methods predicted a higher binding affinity for the GLH-19/siRNA complex. Consistent with these computational results, gel experiments indicated stronger binding and more stable interactions for the GLH-19/siRNA complexes, which in addition showed almost no degradation in the presence of nucleases. cryo-EM studies characterized the bolaamphiphile/siRNA complexes indicating that GLH-19/siRNA and GLH-20/siRNA have different relative hydrophobicities with GLH-19/siRNA being less hydrophobic. Based on cell culture transfections and the in vivo live fluorescence imaging results, we concluded that both GLH-19 and GLH-20 bolaamphiphiles have great potential to be used as carriers for siRNA delivery with GLH-19 being a better candidate. Bola/siRNA complexes significantly increase the chemical stability of siRNAs and provide excellent intracellular uptake followed by specific gene silencing. Moreover, depending on the application, the extent of chemical protection of the siRNA can be altered by simply changing the carrier.