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.

为了控制基于RNA的纳米颗粒的可交付功能和稳定性，将DNA和RNA的性质合并为由RNA/DNA杂交构建的计算设计的纳米颗粒的开发。这些分子允许在血清中较高的稳定性，荧光标记物的附着，用于跟踪而无需干扰RNA功能，以及分裂灭活它们的功能元件的组件的能力，但是在互补的toeholds控制下，可以调节互补的toeholds的激活。 DS siRNA（可固化的底物siRNA）可以分为两个组件，每个组件由RNA/DNA杂种组成，其中DNA包含互补的单链脚趾与其在互补杂交中发现的对应物。当将两种杂种转染到细胞中时，由于脚趾和杂种和产物之间的计算确定的热力学差异，将其重组成两种产物。该产品由及其附着的荧光团诱导的fret效果组成，而另一种产物是DS siRNA，能够使靶向基因沉默。扩展功能扩展到包括多个函数派。孔雀石绿色的适体和DS siRNA被拆分并掺入互补的杂种中。实验表明，在用脚趾重组的链重组后，两种功能都激活了。在另一个实验中，测量了靶向MDA-MB-231/GFP细胞系的含有1-3 ds siRNA的杂种的沉默效率。沉默与杂种中存在的DS siRNA数量成正比，其中3 ds siRNA显示出最佳的沉默。我们表明，使用单链DNA模板，RNA聚合酶II依赖性转录可以产生长裂开功能杂种。诸如LNA之类的转录停止元素的掺入被证明成功地与脚趾产生了混合构建体。测试了I型干扰素反应，结果表明，检测到3 ds siRNA的杂种重新关联的响应最小。但是，由于重组长的DNA链，该响应被证明是由7个组件组成的杂种放置重新定位明显更高的。我们在RNA纳米技术方面的工作引入了新型纳米镜。纳米。除了具有多个不同短的干扰RNA的功能化外，用于组合RNA干扰（例如，针对多个HIV-1基因），纳米含量还允许同时掺入各种RNA适体，荧光染料，蛋白质，蛋白质，蛋白质，RNA-DNA Hybrid，以及RNA-DNA混合型的旨在调节多个分裂功能细胞内部的功能性。我们展示了如何通过掺入针对人表皮生长因子受体的RNA适体来实现细胞靶向特性的方法。同样，由于将RNA功能（例如DS RNA）掺入纳米尺度中，固态化学合成的困难通常不能超过60个核苷酸，因此我们通过使用单股to toe toe toe toe toe toe toe toe toe toe toe toe toe toe toe toe nanoscaffors将DS RNA退化为纳米镜。最后，我们展示了如何通过使用RNA-DNA杂种触发Nanoring的治疗功能。这项新技术涉及将不同的功能分解在RNA-DNA纳米纳的和同源RNA-DNA杂交之间，并与这些功能的条件细胞内激活。使用各种生化，生物物理，体外和体内方法来表征和显示这些颗粒的功效。这包括击倒艾滋病毒，以及在Xenographhemaghosh小鼠模型中的基因沉默。重要的是，干扰素和促炎性细胞因子激活测定法表明，与RNA相比，对DNA纳米颗粒的反应明显较低，这表明这些分子在治疗用途中的潜力更大。我们使用先前表征的链链RNA纳米管作为控制多个siRNA的控制的支架。用六个DS RNA官能化RNA纳米接管。制作了另外两个版本的立方体；一个由带有RNA-DNA杂交DS RNA臂的RNA核心组成，另一个由带有RNA-DNA混合DS RNA ARM的DNA核心组成。后两个病例中的手臂包含DNA脚趾，当带有同源RNA-DNA杂交双链体时，允许功能性siRNA激活。转染实验显示功能激活，包括降低HIV的调节。结果表明，DNA核心立方体的干扰素响应最少，而所有RNA立方都具有最多的响应，而RNA核心立方体位于中间。由于RNA是一个柔性分子，因此重要的是考虑与RNA纳米构建体的自组装有关的后果。由于MD在计算上是耗时的，因此我们探索了粗粒技术，各向异性网络建模（ANM）的使用，可以改变分子表示的粗糙度，从每个核苷酸的1个珠子到完整的原子代表，从每个原子的1个珠子。可以通过将弹簧常数分配给位于每个珠的定义范围内的相互作用来得出力和势能。这种方法缩短了一个通常需要数周的MD到几个小时的模拟。我们专注于低频C运动，作为研究分子最相关的动态特征的指标。我们的纳米管以ANM为特征，结果使计算和实验结果一致。 ANM还深入了解了立方体变体的观察到的组装屈服及其融化温度。我们使用MD，结构特性，均方根偏差，回旋的半径和RNA纳米管的半径半径（RDF），在物理学中的大小至约20Nm。表征了在特定温度下，管子周围的离子浓度是时间的函数。我们发现，当温度升高时，离子的数量在管的一定距离内增加。同样，在管子周围的距离内的离子数减少了。在RNA纳米范围内，RDF图也显示出与温度的类似趋势。基于RNA的纳米构建物在细胞培养和体内的递送对于使用这些药物的治疗方法开发至关重要。非修饰的裸rNA由于核酸酶的血清半衰期短，由于其固有的负电荷而难以越过细胞膜。为了应对其中一些问题，我们评估了氧气醚脂质（OEL），其中包含疏水结构域中的修饰和亲水性头部组，以与siRNA分子和所得复合物的siRNA递送效率进行复合形成。分别使用MDA-MB-231和MDA-MB-231/GFP细胞分析了OELS输送核酸和沉默的潜力。我们发现，将羟基引入到OEL的极性结构域，而不饱和疏水结构域则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的纳米颗粒的简单，有效的组装测定。跟踪RNA组件（例如天然聚丙烯酰胺凝胶和原子力显微镜）的常见方法通常是时间密集型的。我们开发了一种技术，用于使用荧光银纳米簇（AG NC）的形成来快速分析RNA NP组装阶段。该方法利用了Ag NC形成的单链特异性和序列依赖性，以在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写在上述主题上。