Computational and Experimental RNA Nanobiology

计算和实验 RNA 纳米生物学

• 批准号: 8937941
• 负责人: Bruce Shapiro
• 金额: $ 82.08万
• 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/8937941
• 关键词: A549; Accounting; Address; Adenocarcinoma Cell; Affect; Affinity; Agreement; Behavior; Binding; Biocompatible Materials; Biodistribution; Blood; Blood - brain barrier anatomy; Breast Cancer Cell; Cancer cell line; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Cereals; Characteristics; Charge; Complex; DNA; Detection; Development; Digestion; Dimensions; Disease; Dissociation; Elements; Environment; Experimental Designs; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Fluorescence-Activated Cell Sorting; Frequencies; GAG Gene; Gel; Gene Targeting; Genetic; Genetic Recombination; Genetic Transcription; Glutathione S-Transferase; HIV-1; Head; Hela Cells; Hour; Hybrids; Injection of therapeutic agent; Interferon Type I; Kinetics; Lung Adenocarcinoma; MDA MB 231; Malignant Neoplasms; Measures; Methodology; Methods; Micelles; Modeling; Molecular; Molecular Conformation; Motion; Nucleotides; Nude Mice; Organ; Organ Weight; Paper; Peptide Hydrolases; Potential Energy; Production; Property; Protein Binding; RNA; RNA Polymerase II; RNA Stability; RNA-Directed DNA Polymerase; Receptor Cell; Rotation; Serum; Shapes; Side; Single-Stranded DNA; Small Interfering RNA; Stretching; Structure; Tail; Techniques; Temperature; Test Result; Testing; Therapeutic; Thermodynamics; Time; Transfection; Variant; Veins; Vesicle; Weight; Writing; Xenograft procedure; aptamer; arm; base; catalyst; design; flexibility; fluorophore; in vivo; insight; melting; molecular dynamics; mouse model; nanobiology; nanodevice; nanoparticle; network models; nuclease; prevent; programs; reconstitution; research study; response; self assembly; simulation; study characteristics; synthetic biology; therapeutic development; tumor; uptake; yeast two hybrid system

To achieve a greater degree of 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, permitted the attachment of fluorescent markers for tracking without interfering with RNA functionality, and permitted 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 fine-tuned. For example, a 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 presence of 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. GFP was targeted in a breast cancer cell line. The protease and envelope encoding regions were targeted in HIV-1 infected HeLa cells showing significantly decreased GAG production and Reverse Transcriptase activity. In addition, glutathione S-transferase P1 was targeted and down regulated in A549 lung adenocarcinoma cells. In vivo, xenograft MDA-MB-231/GFP tumor mouse models were investigated. Biodistribution, nuclease digestion, and GFP silencing all showed the efficacy of the hybrid delivery methodology described here. Most hybrid constructs were found in the tumor by organ weight, the hybrids lasted longer in the blood than standard siRNAs and GFP silencing was observed after intratumoral injections of the hybrids. The split functionality concept was also applied to the malachite green aptamer. Again, the aptamer became functional after the two halves recombined. The split hybrid functionality was extended to include multiple functionalites in the hybrid constructs. For example, a malachite green aptamer and DS siRNAs were split and incorporated in complementary hybrids. Experiments showed activation of both functionalities upon recombination of the hybrid strands with toeholds. In another experiment the silencing efficiency of hybrids containing 1, 2 and 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 also 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 desired toeholds. Type I interferon response was also 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. Since RNA is inherently a flexible molecule it is important to consider the ramifications related to self-assembly of RNA nanoconstructs that such a characteristic might impose. Modeling of an RNA tectosquare ring using our program RNA2D3D indicated that closure of the ring could be obtained if one arm from each corner of the L-shaped corner motif underwent a 22 degree coaxial rotation. Using molecular dynamics simulations (MD) we showed that such twisting and bending behavior was very possible and could therefore physically account for the closure of the square ring. A computational search methodology was used to search for dynamic structures derived from the MD to close the ring. NanoTiler was also used, with its built-in distortion functions which enabled helical bending, twisting, compression and stretching to close the ring. Since MD is inherently 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 ultimately potential energies can be derived by assigning a spring constant to interactions that lie within a defined range of each bead. Eigenvalues and eigenvectors derived from the interaction matrix are used to determine the frequencies and directions of motions. This approach shortens a simulation that would normally take weeks with MD to just a few hours. We focused on the low frequency collective motions as an indicator of the most biologically relevant dynamic characteristics of the studied molecule. Our nanocubes were characterized with ANM, and the results brought the computational and the experimental results into agreement. ANM results also added insight into the observed assembly yields of the cube variants and their melting temperatures. 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 have been experimenting with bolaamphiphiles (bolas). Bolas consist of 1 or 2 positively charged head groups on each side of a hydrophobic chain. More specifically this study addressed the computational and experimental characterization of two bolas, GLH-19 and GLH-20. They can assemble into either micelles or vesicles, can deliver cargo in a relatively safe and efficient manner, and are capable of crossing the blood-brain barrier. We focused on understanding the molecular basis of the interactions of GLH-19 and GLH-20 micelles with DS siRNA using computational and experimental methods. The differences found could be attributed to the distances of the head groups from the center of the micelles, as determined by molecular dynamics simulations of micelle formation. GLH-20 head groups were more deeply buried. MD revealed that GLH-19 had higher binding affinity with the DS siRNA which correlated to nuclease digestion and gel experiments indicating the same. Cryo-EM results showed micelle formation of both bolas with and without RNA and their sizes were comparable with DLS experiments. It also indicated that GLH-20 was more hydrophobic than GLH-19. Confocal fluorescence microscopy and fluorescence-activated cell sorting (FACS) showed significant cellular uptake with slightly higher efficiency for the GLH-19/DS siRNA micelle complexes. The lesser uptake for the GLH-20/DS siRNA complexes could be explained due to the relatively lower binding affinity of GLH-20 which may promote partial dissociation of the complexes in the transfection media, thus preventing some fraction of the DS siRNAs from entering the cells. However, silencing of GFP in the MDA-MB-231/GFP breast cancer cell line was comparable, thus indicating that GLH-20, due to its lower affinity for the DS siRNA, released the DS siRNA in a more efficient manner. We also demonstrated via in vivo experiments with athymic nude mice with xenograft MDA-MB-231/GFP tumors that organ uptake of tail vein injected DS siRNA's had significantly higher tumor uptake of the DS siRNA in the tumor normalized by weight compared to other organs. Comparable good uptake and silencing was found when the bolas were used in conjunction with our hybrid RNA/DNA duplex experiments. Invited review papers were also written on the above described subject.

为了更大程度地控制基于 RNA 的纳米粒子的可传递功能和稳定性，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，允许在不干扰 RNA 功能的情况下附着荧光标记进行追踪，并允许分裂功能元件的成分，使它们失活，但允许随后在互补立足点的控制下激活，重新结合的动力学可以进行微调。例如，DS siRNA（Diceable 底物 siRNA）可以分为两个组件，每个组件由 RNA/DNA 杂合体组成，其中 DNA 包含与互补杂合体中发现的对应物互补的单链立足点。由于立足点的存在以及计算确定的杂交体和产物之间的热力学差异，当转染到细胞中时，这两种杂交体重组成两种产物。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。 GFP 被靶向乳腺癌细胞系。 HIV-1 感染的 HeLa 细胞中的蛋白酶和包膜编码区被靶向，显示 GAG 产生和逆转录酶活性显着降低。此外，谷胱甘肽S-转移酶P1在A549肺腺癌细胞中被靶向并下调。在体内，研究了异种移植 MDA-MB-231/GFP 肿瘤小鼠模型。生物分布、核酸酶消化和 GFP 沉默均显示了此处描述的混合递送方法的功效。大多数杂交构建体按器官重量在肿瘤中发现，杂交体在血液中的持续时间比标准 siRNA 更长，并且在肿瘤内注射杂交体后观察到 GFP 沉默。分裂功能概念也应用于孔雀石绿适体。两半重新组合后，适体再次发挥作用。分离混合功能被扩展以包括混合结构中的多个功能。例如，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，杂合链与立足点重组后，两种功能都会被激活。在另一项实验中，测量了含有 1、2 和 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。我们还表明，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，LNA 等转录终止元件的掺入可以成功地生成具有所需立足点的杂合构建体。还测试了 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。由于 RNA 本质上是一种灵活的分子，因此必须考虑这种特性可能带来的与 RNA 纳米结构自组装相关的后果。使用我们的程序 RNA2D3D 对 RNA tectosquare 环进行建模表明，如果 L 形角图案每个角的一个臂经历 22 度同轴旋转，则可以获得环的闭合。通过分子动力学模拟（MD），我们表明这种扭曲和弯曲行为是非常有可能的，因此可以从物理上解释方环的闭合。使用计算搜索方法来搜索从 MD 导出的动态结构以闭合环。还使用了 NanoTiler，其内置的扭曲功能可以实现螺旋弯曲、扭曲、压缩和拉伸来闭合环。由于 MD 本质上计算耗时，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 个珠子更改为从 1 个珠子的完整原子表示每个原子。通过为每个珠子的定义范围内的相互作用分配弹簧常数，可以得出力和最终的势能。从交互矩阵导出的特征值和特征向量用于确定运动的频率和方向。这种方法将通常需要数周的 MD 模拟时间缩短为几个小时。我们专注于低频集体运动，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 结果还进一步了解了观察到的立方体变体的组装产量及其熔化温度。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，我们一直在用双亲双亲物（bolas）进行实验。 Bolas 由位于疏水链两侧的 1 或 2 个带正电的头部基团组成。更具体地说，这项研究解决了两个流星 GLH-19 和 GLH-20 的计算和实验特征。它们可以组装成胶束或囊泡，可以以相对安全和有效的方式递送货物，并且能够穿过血脑屏障。我们重点利用计算和实验方法了解 GLH-19 和 GLH-20 胶束与 DS siRNA 相互作用的分子基础。发现的差异可能归因于头基与胶束中心的距离，这是通过胶束形成的分子动力学模拟确定的。 GLH-20头部组埋得更深。 MD揭示GLH-19与DS siRNA具有更高的结合亲和力，这与核酸酶消化和凝胶实验相关，表明同样的情况。冷冻电镜结果显示，有和没有 RNA 的两种球团均形成胶束，其大小与 DLS 实验相当。它还表明GLH-20比GLH-19更具疏水性。共聚焦荧光显微镜和荧光激活细胞分选 (FACS) 显示 GLH-19/DS siRNA 胶束复合物的细胞摄取显着，且效率稍高。 GLH-20/DS siRNA 复合物的摄取较少可以解释为 GLH-20 的结合亲和力相对较低，这可能促进转染介质中复合物的部分解离，从而阻止部分 DS siRNA 进入转染介质。细胞。然而，MDA-MB-231/GFP乳腺癌细胞系中GFP的沉默是可比的，因此表明GLH-20由于其对DS siRNA的亲和力较低，以更有效的方式释放DS siRNA。我们还通过对具有异种移植MDA-MB-231/GFP肿瘤的无胸腺裸鼠的体内实验证明，与其他器官相比，尾静脉注射的DS siRNA的器官摄取在按重量归一化的肿瘤中具有显着更高的DS siRNA的肿瘤摄取。当bolas与我们的杂交RNA/DNA双链体实验结合使用时，发现了相当好的吸收和沉默。还就上述主题撰写了特邀评论论文。