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更高的稳定性，允许附着荧光标记物进行跟踪而无需干扰RNA功能，并允许将功能元素的组成部分分裂为失活的功能元素的组成部分，但是以后的激活能够控制互补的toeholds的控制，重新构成的动力学可以通过这种方式进行了微调。例如，DS siRNA（可滴定的底物siRNA）可以分为两个组件，每个组件由RNA/DNA杂交组成，其中DNA包含与互补杂交中的互补的单链脚趾相互补的单链脚趾。由于脚趾的存在以及杂种和产物之间的计算确定的热力学差异，这两种杂种将转染到细胞中重组成两种产物。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。 GFP针对乳腺癌细胞系。在HIV-1感染的HELA细胞中，蛋白酶和包膜编码区域的靶向显着降低了GAG的产生和逆转录酶活性。此外，在A549肺腺癌细胞中靶向谷胱甘肽S-转移酶P1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沉默都显示了此处描述的混合递送方法的功效。大多数杂化构建体是通过器官重的肿瘤中发现的，杂种在血液中的持续时间比标准siRNA的持续时间更长，并且在肿瘤内注射杂种后观察到GFP沉默。分裂功能概念也应用于孔雀石绿色适体。同样，在两半重组后，适体开始起作用。扩展了分裂的杂种功能，以在混合构建体中包括多个功能。例如，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，在杂种链与脚趾的重组后，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、2和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。我们还表明，使用单链DNA模板可以由RNA聚合酶II依赖性转录产生长的分裂功能杂种。诸如LNA之类的转录停止元素的掺入被证明成功地与所需的脚趾产生了混合构造。还测试了I型干扰素反应，结果表明，检测到3 ds siRNA的杂化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。由于RNA固有地是一个柔性分子，因此重要的是考虑与这种特征可能施加的RNA纳米结构的自组装相关的后果。使用我们的程序RNA2D3D对RNA TectoSquare环进行建模表明，如果从L形角基序的每个角落的一个臂进行了22度同轴旋转，则可以获得环的闭合。使用分子动力学模拟（MD），我们表明这种扭曲和弯曲行为非常可能，因此可以物理地解释方形环的闭合。使用计算搜索方法来搜索从MD衍生的动态结构以关闭环。还使用了纳米动物的内置失真功能，可实现螺旋弯曲，扭曲，压缩和拉伸以关闭环。由于MD本质上是计算时间耗时的，因此我们探索了粗粒技术，各向异性网络建模（ANM）的使用，可以改变分子的表示从每个核苷酸的1个珠子到每个原子的1个珠子的完整原子表示。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最终的势能。从相互作用矩阵得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。我们专注于低频集体运动，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结果还增加了对观察到的立方体变体的组装屈服及其熔融温度的洞察力。细胞培养和体内基于RNA的纳米构建物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，我们一直在尝试Bolaamphiphiles（Bolas）。 Bolas由疏水链每一侧的1或2个带正电荷的头部组组成。更具体地说，这项研究探讨了两个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，可以以相对安全有效的方式运送货物，并能够越过血脑屏障。我们专注于使用计算和实验方法来理解GLH-19和GLH-20胶束与DS siRNA的相互作用的分子基础。所发现的差异可以归因于胶束形成的分子动力学模拟确定的头部距离胶束中心的距离。 GLH-20头组被更深入埋葬。 MD显示，GLH-19与DS siRNA具有较高的结合亲和力，该亲和力与核酸酶消化和凝胶实验相关。冷冻EM结果表明，有和没有RNA的两个带有和没有RNA的Bolas的胶束形成与DLS实验相当。这也表明GLH-20比GLH-19更疏水。共聚焦荧光显微镜和荧光激活的细胞分选（FACS）显示出明显的细胞摄取，对于GLH-19/DS siRNA胶束复合物的效率略高。 GLH-20/DS siRNA复合物的较小摄取可以解释，因为GLH-20的结合亲和力相对较低，这可能会促进转染介质中络合物的部分解离，从而阻止DS siRNA的一部分进入细胞。然而，GFP在MDA-MB-231/GFP乳腺癌细胞系中的沉默是可比的，因此表明GLH-20由于其对DS siRNA的较低亲和力而以更有效的方式释放了DS siRNA。我们还通过与异种移植物MDA-MB-231/GFP肿瘤进行的无性裸鼠体内实验证明，与其他器官相比，在肿瘤中，DS siRNA的尾静脉注射DS siRNA的肿瘤吸收较高的肿瘤肿瘤的肿瘤吸收明显更高。当与我们的混合RNA/DNA双链实验结合使用Bolas时，发现了可比的良好摄取和沉默。邀请的评论论文也写在上述主题上。