Computational and Experimental RNA Nanobiology

为了控制基于RNA的纳米颗粒的可传递功能和稳定性，在开发由RNA/DNA杂交构建的计算设计纳米颗粒时，融合了DNA和RNA的特性。这些分子在血清中具有更高的稳定性，在不干扰RNA功能的情况下附着荧光标记进行跟踪，并且能够分裂功能元件的成分，使其失活，但允许在互补支点的控制下稍后激活，通过互补支点可以调整重新结合的动力学。DS siRNA（可切割底物siRNA）可以分成两部分，每一部分都由RNA/DNA杂交体组成，其中DNA包含互补的单链支点，与互补杂交体中的对应物互补。这两个杂交种，当转染到细胞中，由于结点和计算确定的杂交种和产物之间的热力学差异，重新组合成两个产物。其中一种产物由DNA双链及其附着的荧光团组成，可诱导FRET效应，而另一种产物是能够沉默目标基因的DS siRNA。拆分功能被扩展为包含多个功能。将孔雀石绿适体和DS sirna分离并结合到互补杂种中。实验表明，这两种功能的激活后重组链与脚。在另一项实验中，我们测量了含有1-3个DS sirna的杂合体靶向MDA-MB-231/GFP细胞系的沉默效率。沉默与存在的DS siRNA数量成正比，其中3个DS siRNA沉默效果最好。我们发现，利用单链DNA模板，通过RNA聚合酶ii依赖性转录，可以产生长分裂功能杂交体。转录停止元件（如LNAs）的结合被证明成功地产生了具有支点的杂交结构。对I型干扰素反应进行了测试，结果表明，对3ds sirna的杂交再关联检测到最小的反应。然而，对于由7个组分组成的杂交重组，由于重组的是长DNA链，因此反应明显更高。我们在RNA纳米技术方面的工作引入了新的纳米支架，例如纳米结构。除了用多种不同的短干扰RNA进行组合RNA干扰（例如，针对多个HIV-1基因）的功能化外，纳米环还允许同时掺入各种RNA适体、荧光染料、蛋白质以及RNA- dna杂交体，目的是有条件地激活细胞内的多种分裂功能。我们展示了纳米化设计如何通过结合人类表皮生长因子受体特异性的RNA适体来实现细胞靶向特性。此外，由于将RNA功能（如DS RNA）整合到纳米支架中会给固体化学合成带来困难，因为RNA成分的长度通常不超过60个核苷酸，因此我们通过使用单链脚位将DS RNA退火到纳米支架中来解决这个问题。最后，我们展示了纳米环的治疗功能如何通过使用RNA-DNA杂交体来触发。这项新技术涉及在RNA-DNA纳米环和同源RNA-DNA杂交体之间分裂不同的功能，并有条件地在细胞内激活这些功能。各种生物化学、生物物理、体外和体内方法被用来表征和显示这些颗粒的功效。这包括敲除HIV，以及在xenograph小鼠模型中沉默基因。重要的是，干扰素和促炎细胞因子激活试验表明，与RNA相比，DNA纳米颗粒的反应明显较低，这表明这些分子在治疗用途上具有更大的潜力。我们使用先前表征的六链RNA纳米立方体作为多个sirna的受控递送的支架。RNA纳米立方被6个DS RNA功能化。另外两个版本的立方体被制作出来；一种由带有RNA-DNA杂交DS RNA臂的RNA核组成，另一种由带有RNA-DNA杂交DS RNA臂的DNA核组成。后两种情况下的臂含有DNA支点，当呈现同源RNA-DNA杂交双工时，它允许功能性siRNA激活。转染实验显示激活功能包括下调HIV。结果表明，DNA核立方体对干扰素的响应最小，RNA核立方体对干扰素的响应最大，RNA核立方体居中。由于RNA是一种柔性分子，因此考虑与RNA纳米结构的自组装相关的分支是很重要的。由于MD在计算上很耗时，我们探索了一种粗粒度技术的使用，即各向异性网络建模（ANM），它可以改变分子表示的粗程度，从每个核苷酸1个头到每个原子1个头。力和势能可以通过在每个头的定义范围内指定一个弹簧常数来推导。这种方法将MD通常需要数周的模拟时间缩短到几个小时。我们将重点放在低频c运动上，作为所研究分子最具生物学相关性的动态特性的指标。用ANM对我们的纳米立方体进行了表征，计算结果与实验结果吻合。ANM还增加了对观察到的立方体变体的组装产量及其熔化温度的见解。利用分子动力学研究了20nm左右的RNA纳米管在生理溶液中的结构性质、均方根偏差、旋转半径和径向分布函数（RDF）。研究了在特定温度下，管周围离子浓度随时间的变化规律。我们发现，当温度升高时，在管的一定距离内离子的数量增加。此外，在管周围这个距离内的离子数量在淬火运行中减少。在RNA纳米化的情况下，RDF图也显示了与温度相似的趋势。基于rna的纳米结构物在细胞培养和体内的传递对于使用这些药物的治疗方法的发展至关重要。由于核酸酶的作用，未修饰的裸rna在血清中的半衰期较短，并且由于其固有的负电荷而难以穿过细胞膜。为了解决这些问题，我们评估了肟醚脂质（OELs）在疏水结构域和亲水头基中含有修饰，用于与siRNA分子形成复合物，以及由此产生的复合物的siRNA递送效率。利用MDA-MB-231和MDA-MB-231/GFP细胞分别分析了OELs传递核酸和沉默绿色荧光蛋白基因的潜力。我们发现，在细胞培养中，在OELs的极性区域引入羟基和在疏水区域不饱和有利于更高的转染和基因沉默。目前需要一种简单、高效的rna纳米颗粒组装测定方法。常用的方法来跟踪RNA组装，如天然聚丙烯酰胺凝胶和原子力显微镜往往是耗时的。我们开发了一种利用荧光银纳米团簇（Ag NC）的形成快速分析RNA NP组装阶段的技术。该方法利用Ag NC形成的单链特异性和序列依赖性，为RNA NP组装的每个阶段产生独特的光学读数。邀请的评论论文和书籍章节也撰写了上述主题。

To achieve control over deliverable functionality and stability of RNA-based nanoparticles, the properties of DNA and RNA were merged in the development of computationally designed nanoparticles that were constructed from RNA/DNA hybrids. These molecules allowed higher stability in blood serum, attachment of fluorescent markers for tracking without interfering with RNA functionality, and the ability to split the components of functional elements inactivating them, but allowing later activation under the control of complementary toeholds by which the kinetics of re-association can be tuned. DS siRNAs (Diceable substrate siRNA) could be split into two components, each consisting of an RNA/DNA hybrid, where the DNA contains a complementary single-stranded toehold to its counterpart found in a complementary hybrid. The two hybrids, when transfected into cells recombine into two products due to the toeholds and the computationally determined thermodynamic difference between the hybrids and the products. The products, one consisting of a DNA duplex with its attached fluorophores induced a FRET affect, while the other product was a DS siRNA capable of silencing the targeted gene. The split functionality was extended to include multiple functionalites. A malachite green aptamer and DS siRNAs were split and incorporated in complementary hybrids. Experiments showed activation of both functionalities upon recombination of the strands with toeholds. In another experiment the silencing efficiency of hybrids containing 1-3 DS siRNAs targeting MDA-MB-231/GFP cell lines was measured. Silencing was proportional to the number of DS siRNA present in the hybrid with 3 DS siRNA showing the best silencing. We showed that long split functional hybrids can be produced by RNA polymerase II-dependent transcription using single-stranded DNA templates. The incorporation of transcription stop elements such as LNAs proved successful in generating hybrid constructs with the toeholds. Type I interferon response was tested and the results indicated that a minimal response was detected for hybrid reassociation of 3 DS siRNAs. However, the response was shown to be significantly higher for hybrid reassociations consisting of 7 components due to long DNA strands being reconstituted. Our work in RNA nanotechnology introduced novel nanoscaffolds e.g. nanorings. Besides functionalization with multiple different short interfering RNAs for combinatorial RNA interference (e.g., against multiple HIV-1 genes), nanorings also allow simultaneous incorporation of assorted RNA aptamers, fluorescent dyes, proteins, as well as RNA-DNA hybrids aimed to conditionally activate multiple split functionalities inside cells. We showed how the nanoring design can achieve cell-targeting properties through incorporation of RNA aptamers specific for the human epidermal growth factor receptor. Also, since the incorporation of RNA functionalities such as DS RNAs into the nanoscaffolds presents difficulties for solid state chemical synthesis as RNA components generally cannot exceed 60 nucleotides in length, we solved this problem by annealing DS RNAs to nanoscaffolds using single-stranded toehold sites. Finally we showed how the therapeutic functionality of the nanoring can be triggered by the use of RNA-DNA hybrids. This new technique involves splitting the different functionalities between a RNA-DNA nanoring and cognate RNA-DNA hybrids with conditional intracellular activation of these functionalities. Various biochemical, biophysical, in vitro and in vivo methods were used to characterize and show the efficacy of these particles. This included knock down of HIV, and silencing of genes in xenograph mouse models. Importantly, interferon and pro-inflammatory cytokine activation assays indicated significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use. We used previously characterizedsix-stranded RNA nanocubes as scaffolds for the controlled delivery of multiple siRNAs. The RNA nanocubes were functionalized with six DS RNAs. Two other versions of the cube were made; one consisting of an RNA core with RNA-DNA hybrid DS RNA arms and the other consisting of a DNA core with RNA-DNA hybrid DS RNA arms. The arms in the latter two cases contained DNA toeholds which allowed for functional siRNA activation when presented with cognate RNA-DNA hybrid duplexes. Transfection experiments showed activation of functionality including down regulation of HIV. It was shown that DNA core cubes had the least interferon response, while all RNA cubes had the most, while the RNA core cube was in the middle. Since RNA is a flexible molecule it is important to consider the ramifications of this related to self-assembly of RNA nanoconstructs. Since MD is computationally time-consuming, we explored the use of a coarse-grained technique, Anisotropic Network Modeling (ANM), which can vary the coarseness of a molecule's representation from 1 bead per nucleotide, to a full atomic representation from 1 bead per atom. Forces and potential energies can be derived by assigning a spring constant to interactions that lie within a defined range of each bead. This approach shortens a simulation that would normally take weeks with MD to just a few hours. We focused on the low frequency c motions as an indicator of the most biologically relevant dynamic characteristics of the studied molecule. Our nanocubes were characterized with ANM, and results brought the computational and the experimental results into agreement. ANM also added insight into the observed assembly yields of the cube variants and their melting temperatures.We studied, using MD, the structural properties, Root Mean Square Deviation, the radius of gyration and radial distribution function (RDF) of RNA nanotubes up to the size of about 20nm in physological solutions. The concentration of ions around the tube as a function of time at a particular temperature were characterized. We found that when the temperature increases, the number of ions increased within a certain distance of the tube. Also, the number of ions within this distance around the tube decreases in quenched runs. RDF plots also demonstrated a similar trend with temperature in the case of RNA nanorings.The delivery of RNA-based nanoconstructs in cell culture and in vivo is essential for the development of therapeutic methodologies using these agents. Non-modified naked RNAs have short half-lives in blood serum due to nucleases and have difficulty crossing cell membranes due to their inherent negative charge. To counter some of these issues we evaluated oxime ether lipids (OELs) containing modifications in the hydrophobic domains and hydrophilic head groups for complex formation with siRNA molecules and siRNA delivery efficiency of resulting complexes. The potential of OELs to deliver nucleic acids and silence the green fluorescent protein gene was analyzed using MDA-MB-231 and MDA-MB-231/GFP cells, respectively. We found that the introduction of hydroxyl groups to the polar domain of the OELs and unsaturation into the hydrophobic domain favor higher transfection and gene silencing in a cell cultures. There is a need for simple, efficient assembly assays of RNA-based nanoparticles. Common methods for tracking RNA assemblies such as native polyacrylamide gels and atomic force microscopy are often time-intensive. We developed a technique for rapid analysis of RNA NP assembly stages using the formation of fluorescent silver nanoclusters (Ag NC). This method exploits the single-stranded specificity and sequence dependence of Ag NC formation to produce unique optical readouts for each stage of RNA NP assembly. Invited review papers and book chapters were also written on the above described subjects.