Computational and Experimental RNA Nanobiology

计算和实验 RNA 纳米生物学

• 批准号: 9153759
• 负责人: Bruce Shapiro
• 金额: $ 90.43万
• 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/9153759
• 关键词: Affect; Agreement; Atomic Force Microscopy; Biochemical; Biocompatible Materials; Biological Assay; Book Chapters; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Cereals; Charge; Complex; Cytokine Activation; DNA; Dependence; Detection; Development; Dimensions; Disease; Down-Regulation; Elements; Environment; Epidermal Growth Factor Receptor; Ethers; Experimental Designs; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Frequencies; Gene Silencing; Gene Targeting; Genes; Genetic Recombination; Genetic Transcription; Green Fluorescent Proteins; HIV; HIV-1; Head; Hour; Human; Hybrids; In Vitro; Inflammatory; Interferon Type I; Interferons; Ions; Kinetics; Length; Lipids; MDA MB 231; Malignant Neoplasms; Measures; Methodology; Methods; Modification; Molecular; Molecular Conformation; Motion; Nanotechnology; Nanotubes; Nucleic Acids; Nucleotides; Optics; Oximes; Paper; Plant Roots; Potential Energy; Problem Solving; Property; Protein Binding; RNA; RNA Interference; RNA Polymerase II; RNA Stability; RNA analysis; Radial; Receptor Cell; Running; Serum; Silver; Single-Stranded DNA; Site; Small Interfering RNA; Solutions; Specificity; Staging; Structure; Techniques; Temperature; Test Result; Testing; Therapeutic; Therapeutic Uses; Thermodynamics; Time; Transfection; Tube; Variant; Work; Writing; aerobic respiration control protein; aptamer; arm; base; catalyst; chemical synthesis; combinatorial; design; flexibility; fluorophore; genetic information; hydroxyl group; in vivo; insight; knock-down; melting; mouse model; nanobiology; nanodevice; nanoparticle; network models; novel; nuclease; particle; polyacrylamide gels; rapid technique; reconstitution; research study; response; scaffold; self assembly; simulation; solid state; study characteristics; synthetic biology; therapeutic development; trend; yeast two hybrid system

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.

为了实现对基于 RNA 的纳米粒子的可传递功能和稳定性的控制，DNA 和 RNA 的特性被合并到由 RNA/DNA 杂合体构建的计算设计纳米粒子的开发中。这些分子在血清中具有更高的稳定性，可以在不干扰 RNA 功能的情况下附着荧光标记进行追踪，并且能够分裂功能元件的成分，使其失活，但允许随后在互补立足点的控制下激活，从而重新激活 RNA 的动力学。 -关联可以调整。 DS siRNA（Diceable 底物 siRNA）可以分为两个组件，每个组件均由 RNA/DNA 杂合体组成，其中 DNA 包含与互补杂合体中的对应物互补的单链立足点。当转染到细胞中时，这两种杂交体由于立足点以及计算确定的杂交体和产物之间的热力学差异而重组成两种产物。这些产品，一种由 DNA 双链体及其附着的荧光团组成，诱导 FRET 效应，而另一种产品是能够沉默靶基因的 DS siRNA。拆分功能已扩展为包含多个功能。将孔雀石绿适体和 DS siRNA 分开并掺入互补杂交体中。实验表明，当股线与立足点重组时，两种功能都会被激活。在另一个实验中，测量了含有 1-3 个 DS siRNA 的杂交体的沉默效率，该 siRNA 靶向 MDA-MB-231/GFP 细胞系。沉默与杂交体中存在的 DS siRNA 的数量成正比，其中 3 DS siRNA 显示出最佳沉默。我们证明，使用单链 DNA 模板，可以通过 RNA 聚合酶 II 依赖性转录来产生长分裂功能杂合体。事实证明，转录终止元件（例如 LNA）的掺入可以成功地生成具有立足点的混合结构。测试了 I 型干扰素反应，结果表明检测到 3 DS siRNA 的混合重联反应最小。然而，由于长 DNA 链的重建，由 7 个成分组成的杂合重关联的响应明显更高。我们在 RNA 纳米技术方面的工作引入了新型纳米支架，例如纳米环。除了用多种不同的短干扰 RNA 进行功能化以进行组合 RNA 干扰（例如针对多个 HIV-1 基因）之外，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，旨在有条件地激活多个细胞内的功能分裂。我们展示了纳米环设计如何通过掺入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 DS RNA 等 RNA 功能掺入纳米支架中给固态化学合成带来了困难，因为 RNA 成分的长度通常不能超过 60 个核苷酸，因此我们通过使用单链立足点将 DS RNA 退火到纳米支架上解决了这个问题。最后，我们展示了如何通过使用 RNA-DNA 杂交体来触发纳米环的治疗功能。这项新技术涉及拆分 RNA-DNA 纳米环和同源 RNA-DNA 杂交体之间的不同功能，并在细胞内条件性地激活这些功能。使用各种生物化学、生物物理、体外和体内方法来表征和显示这些颗粒的功效。这包括在异种小鼠模型中抑制艾滋病毒和基因沉默。重要的是，干扰素和促炎细胞因子激活测定表明，与 RNA 对应物相比，DNA 纳米颗粒的反应明显较低，这表明这些分子具有更大的治疗用途潜力。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 纳米立方体用 6 个 DS RNA 进行功能化。立方体的另外两个版本也被制作出来：一种由RNA核心和RNA-DNA混合DS RNA臂组成，另一种由DNA核心和RNA-DNA混合DS RNA臂组成。后两个案例中的臂包含 DNA 立足点，当与同源 RNA-DNA 杂合双链体一起呈现时，允许功能性 siRNA 激活。转染实验显示功能的激活，包括 HIV 的下调。结果表明，DNA 核心立方体的干扰素反应最少，而所有 RNA 立方体的干扰素反应最多，而 RNA 核心立方体处于中间。由于 RNA 是一种灵活的分子，因此必须考虑其与 RNA 纳米结构自组装相关的后果。由于 MD 在计算上非常耗时，因此我们探索了粗粒度技术的使用，即各向异性网络建模 (ANM)，该技术可以将分子表示的粗糙度从每个核苷酸 1 个珠子改变为从每个核苷酸 1 个珠子到完整的原子表示。原子。可以通过为每个珠子的定义范围内的相互作用分配弹簧常数来导出力和势能。这种方法将通常需要数周的 MD 模拟时间缩短为几个小时。我们专注于低频 c 运动，作为所研究分子最具有生物学相关性的动态特征的指标。我们的纳米立方体用 ANM 进行了表征，结果使计算结果和实验结果一致。 ANM 还深入了解了观察到的立方体变体的组装产率及其熔化温度。我们使用 MD 研究了 RNA 纳米管的结构特性、均方根偏差、回转半径和径向分布函数 (RDF)，最高可达在生理溶液中尺寸约为20nm。表征了特定温度下管周围离子浓度随时间的变化。我们发现，当温度升高时，管内一定距离内的离子数量增加。此外，在淬火运行中，管周围该距离内的离子数量也会减少。 RDF 图也显示出 RNA 纳米环随温度变化的类似趋势。在细胞培养物和体内递送基于 RNA 的纳米结构对于开发使用这些药物的治疗方法至关重要。由于核酸酶的影响，未修饰的裸露 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。为了解决其中一些问题，我们评估了含有疏水结构域和亲水头基修饰的肟醚脂质 (OEL)，以与 siRNA 分子形成复合物以及所得复合物的 siRNA 递送效率。分别使用 MDA-MB-231 和 MDA-MB-231/GFP 细胞分析了 OEL 递送核酸和沉默绿色荧光蛋白基因的潜力。我们发现，将羟基引入 OEL 的极性结构域并将不饱和度引入疏水结构域有利于细胞培养物中更高的转染和基因沉默。需要对基于 RNA 的纳米粒子进行简单、有效的组装测定。追踪 RNA 组装的常用方法（例如天然聚丙烯酰胺凝胶和原子力显微镜）通常非常耗时。我们开发了一种利用荧光银纳米簇 (Ag NC) 的形成来快速分析 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。还就上述主题撰写了特邀评论论文和书籍章节。