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.

为了更好地控制基于RNA的纳米颗粒的可交付功能和稳定性，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，允许在不干扰RNA功能的情况下附着荧光标记进行跟踪，并且允许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，通过互补支点可以微调重新结合的动力学。例如，DS siRNA（可切割底物siRNA）可以分成两个部分，每个部分由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，当转染到细胞中重组为两个产物，由于存在的支点和计算确定的杂交种和产物之间的热力学差异。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。GFP在乳腺癌细胞系中被靶向。在HIV-1感染的HeLa细胞中，蛋白酶和包膜编码区显示GAG产生和逆转录酶活性显著降低。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沉默都显示了本文描述的杂交递送方法的有效性。根据器官重量在肿瘤中发现了大多数杂交结构，杂交结构在血液中的持续时间比标准sirna长，并且在肿瘤内注射杂交结构后观察到GFP沉默。拆分功能的概念也应用于孔雀石绿适配体。再一次，适配体在两半重组后开始发挥功能。拆分混合功能被扩展为在混合结构中包含多个功能。例如，孔雀石绿适体和DS sirna被分离并合并到互补杂交中。实验表明，这两种功能的激活与脚的杂交链重组。在另一项实验中，我们测量了含有1、2和3个DS sirna的杂合体靶向MDA-MB-231/GFP细胞株的沉默效率。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。我们还发现，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所需支点的杂交结构。还测试了I型干扰素反应，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。由于RNA本质上是一种灵活的分子，因此考虑这种特性可能带来的RNA纳米结构自组装的后果是很重要的。利用我们的程序RNA2D3D对一个RNA构造方环进行建模，结果表明，如果l形角基序的每个角的一个臂进行22度的同轴旋转，则可以获得环的闭合。利用分子动力学模拟（MD），我们证明了这种扭曲和弯曲行为是非常可能的，因此可以从物理上解释方形环的闭合。采用计算搜索方法搜索由MD导出的动力结构来闭合环。NanoTiler也被使用，其内置的扭曲功能可以通过螺旋弯曲、扭曲、压缩和拉伸来关闭环。由于MD固有的计算耗时，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，最终势能可以通过在每个头的一定范围内指定一个弹簧常数来推导。由相互作用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集体运动上，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结果还增加了对观察到的立方体变体的组装产率及其熔化温度的见解。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，我们一直在试验亲bolaamphiphiles （bolas）。Bolas由疏水链两侧的1或2个带正电的头基组成。更具体地说，本研究解决了两个bolas GLH-19和GLH-20的计算和实验表征。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运送货物，并且能够穿过血脑屏障。我们主要通过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。发现的差异可归因于头基团与胶束中心的距离，这是由胶束形成的分子动力学模拟确定的。GLH-20头组埋得更深。MD结果显示GLH-19与DS siRNA具有较高的结合亲和力，这与核酸酶酶切相关，凝胶实验也表明了这一点。cro - em结果显示，含RNA和不含RNA的bolas均形成胶束，其大小与DLS实验相当。GLH-20比GLH-19具有更强的疏水性。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效率略高。GLH-20/DS siRNA复合物的摄取较少可能是由于GLH-20的结合亲和力相对较低，这可能会促进复合物在转染介质中的部分解离，从而阻止部分DS siRNA进入细胞。然而，在MDA-MB-231/GFP乳腺癌细胞系中，GFP的沉默是相似的，这表明GLH-20由于对DS siRNA的亲和力较低，以更有效的方式释放DS siRNA。我们还通过胸腺裸鼠移植MDA-MB-231/GFP肿瘤的体内实验证明，与其他器官相比，尾静脉注射DS siRNA的器官摄取在按重量归一化的肿瘤中具有显著更高的DS siRNA的肿瘤摄取。当bolas与我们的杂交RNA/DNA双工实验结合使用时，发现同样良好的吸收和沉默。邀请的评论论文也写在上述主题。