Computational and Experimental RNA Nanobiology

为了控制基于RNA的纳米颗粒的可传递功能和稳定性，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，可以附着荧光标记物进行跟踪，并且能够分离功能元素的成分，使其失活，但在互补支点的控制下允许稍后激活，通过互补支点可以调整重新结合的动力学。DS siRNA（可切割底物siRNA）可以分为两个组分，每个组分由RNA/DNA杂交组成。现在互补的RNA单链支点而不是DNA被用于杂交的构建。这两个杂交种，当转染到细胞中，由于结点和计算确定的杂交种和产物之间的热力学差异，重新组合成两个产物。先前设计的携带6个杂交体的RNA环支架现在包含RNA支点而不是DNA。研究了2、4、6和8个核苷酸的位点的再结合效率。FRET显示，更长的脚导致提高再结合率。这些颗粒的功能在不同的人类细胞系中得到证实。从热力学的角度来看，RNA支点的使用是有利的，因为它减少了解压缩杂合体和产生功能性RNA元件所需的单链末端的长度。从设计的角度来看，RNA支点可以是功能性DS RNA的一部分，或其他潜在的RNA片段，从而减小了所产生的杂交双工的大小并最大限度地减少了设计限制。含有ssRNA支点的条件杂交体也被证明有利于融入更复杂的RNA纳米颗粒。研究表明，与dna -趾基杂合物功能化的RNA纳米粒相比，与RNA -趾基杂合物功能化的RNA纳米粒在酶促共转录合成中表现出更高的产量，并且总体纳米粒尺寸减小。另外一个方案也利用RNA-RNA相互作用。在这里，RNA链被设计成与细胞中特定的mRNA链相互作用。RNA链包含“治疗”和“触发”两种成分，它们被设计成在“触发”mRNA存在的情况下相互分离，形成一种副产物，以及一种短的发夹状RNA，这种RNA可以被切粒酶加工成功能性siRNA。构象变化的发生是由于在“触发”结合链中存在一个扩展的ssRNA立足点，它允许与病变细胞中的mRNA特异性结合。这种方法允许治疗性rna的条件激活，只有在指定的触发链存在。对于治疗癌症等疾病的潜在用途，这可以减少脱靶沉默，并允许更精确的治疗。由于RNA复合物的自组装是几条RNA链的相互作用，一种新的算法HyperFold被开发出来，它可以预测所有可能的链复合物组合的折叠特性。此外，寻找最低自由能碱基配对的一般问题对于计算算法来说是困难的。由于可能的碱基对组合的数量随着序列长度呈指数增长，对于具有长链和假结结构的核酸结构，搜索所有可能的碱基对组合很快变得令人望而却步。HyperFold使用依赖于单个参数的可调启发式解决了这个问题。默认情况下，搜索使用“中间立场”策略。用于预测可能具有复杂“结”结构的多条RNA和DNA链之间的相互作用。基于已知的通过连接子螺旋连接的3D结构元素设计自组装RNA环结构是一项具有挑战性的任务，因为大量的基序组合，其中许多不会导致环闭合。我们通过RNA 3-way结、凸起和亲吻环的组合组装，并将导致环形成的情况制成表格，开发了一种计算机解决方案来解决这个设计问题。找到的解决方案以网络环形目录的形式提供。作为该资源潜在用途的一个例子，我们选择了一个由五条RNA链组成的预测RNA方形结构，并通过实验证明，这五条RNA链的自组装导致方形复合体的形成。一个新的概念是利用同源的核酸纳米粒子，它们是完全互补和相互依赖的功能，其中设计的纳米粒子组之间的物理相互作用启动一个快速的等温形状变化，进而触发多种功能和生物途径的激活，包括转录，能量转移，功能适体和RNA干扰。单个纳米颗粒不具有活性，具有可控制的再结合动力学和可微调的化学和热力学稳定性。此外，纳米颗粒可调节的免疫刺激特性表明，不诱导促炎细胞因子和高水平干扰素的颗粒可以用作携带治疗性寡核苷酸的支架，而具有强干扰素和促炎细胞因子诱导的颗粒可能有资格作为疫苗佐剂。这种双同源纳米粒子方法是我们之前工作的产物，我们用RNA或DNA核心构建了可激活的混合立方体，每个立方体都有自己的可控免疫反应。由于我们能够控制免疫反应，我们已经开始了两个新的合作（Joost Oppenheim- CCR和Chris Jewell， UMD）利用这些特性来激活免疫系统进行抗癌治疗。此外，我们一直在与（Electron Kebebew，临床中心，NCI）合作，使用我们基于rna的纳米颗粒靶向一种致命形式的甲状腺癌。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，我们一直在研究各种脂质和聚合物配方。在脂质方面，我们构建了含有透明质酸的递送剂，用于靶向具有CD44受体的癌细胞（与Esta Sterneck， CCR合作）。最初的实验看起来相当积极。此外，我们还与Jonathan Lovell（布法罗大学）合作开发光活化聚合物，用于递送我们的rna纳米颗粒。初步结果也相当令人鼓舞。一本关于RNA纳米生物学协议的特邀书正在出版中，此外，还撰写了几篇关于上述主题的特邀评论论文和书籍章节。

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 allow higher stability in blood serum, attachment of fluorescent markers for tracking, 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. Now complementary RNA single-stranded toeholds rather than DNA are used in the construction of the hybrids. 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. Previously designed RNA ring scaffolds carrying six hybrids, now contain RNA toeholds rather than DNA. Toeholds of 2, 4, 6, and 8 nucleotides were investigated for their efficiency of re-association. FRET revealed that longer toeholds led to improved re-association rates. The functionality of these particles was confirmed in different human cell lines. From the perspective of thermodynamics, the use of RNA toeholds is advantageous as it reduces the length of the single stranded ends required to unzip the hybrids and generate the functional RNA element. From a design perspective, the RNA toehold can be part of the functional DS RNA, or other potential RNA moiety, reducing the size and minimizing the design constraints of the resulting hybrid duplexes. Conditional hybrids that contain ssRNA toeholds also prove advantageous for incorporation into more complex RNA nanoparticles. It was shown that RNA nanorings functionalized with RNA toeholded hybrids exhibited increased yields from enzymatic co-transcriptional synthesis, as well as reduced overall nanoparticle size, compared to nanorings functionalized with DNA-toeholded hybrid duplexes. An additional scheme has also been exploited using RNA-RNA interactions. Here, an RNA strand is designed to interact with specific mRNA strands in cells. The RNA strand contains both "therapeutic" and "trigger" components that are designed to dissociate from each other in the presence of a "trigger" mRNA and form a byproduct as well as a short hairpin-like RNA which can be processed by dicer to form functional siRNA. The conformational change takes place due to the presence of an extended ssRNA toehold in the "trigger"-binding strand which allows for the specific binding to an mRNA in diseased cells. This approach allows for the conditional activation of therapeutic RNAs only where a designated trigger strand is present. For potential use in treatment of diseases, such as cancer, this can reduce off target silencing and allow for more precise treatment. Because the self-assembly of RNA complexes is an interplay of several RNA strands, a new algorithm, HyperFold was developed that predicts the folding properties of all possible combinations of strand complexes. Also, the general problem of finding the lowest free energy base pairing is difficult for computational algorithms. Because the number of possible base pair combinations grows exponentially with sequence length, searching through all possible base pair combinations quickly becomes prohibitive for nucleic acid structures possessing long strands and pseudoknotted structures. HyperFold solves this problem using a tunable heuristic that depends on a single parameter. By default the search utilizes a "middle-ground" strategy. for predicting the interactions between multiple RNA and DNA strands with possibly complex "knotted" structures. Designing self-assembling RNA ring structures based on known 3D structural elements connected via linker helices is a challenging task due to the immense number of motif combinations, many of which do not lead to ring-closure. We developed an in silico solution to this design problem by combinatorial assembly of RNA 3-way junctions, bulges, and kissing loops, and tabulating the cases that lead to ring formation. The solutions found are made available in the form of a web Ring Catalog. As an example of a potential use of this resource, we chose a predicted RNA square structure consisting of five RNA strands and demonstrated experimentally that the self-assembly of those five strands leads to the formation of a square-like complex. A new concept was developed that utilizes cognate nucleic acid nanoparticles which are fully complementary and functionally-interdependent to each other, whereby the physical interaction between sets of designed nanoparticles initiates a rapid isothermal shape change which in turn triggers the activation of multiple functionalities and biological pathways including transcription, energy transfer, functional aptamers and RNA interference. The individual nanoparticles are not active and have controllable kinetics of re-association and fine-tunable chemical and thermodynamic stabilities. Additionally, tunable immunostimulatory properties of the nanoparticles suggest that the particles that do not induce pro-inflammatory cytokines and high levels of interferons can be used as scaffolds to carry therapeutic oligonucleotides, while particles with strong interferon and proinflammatory cytokine induction may qualify as vaccine adjuvants.This dual cognate nanoparticle approach is an outgrowth of our previous work with the activatable hybrid cubes built from RNA or DNA cores with hybrid arms each cube having its own controllable immune responses. Since we are able to control immune response, we have embarked on 2 new collaborations (Joost Oppenheim- CCR, and Chris Jewell, UMD) to take advantage of these properties to activate the immune system for anti-cancer treatment. In addition, we have been working with (Electron Kebebew, Clinical Center, NCI) on the use of our RNA-based nanoparticles to target a lethal form of thyroid cancer. 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 working with various lipid and polymer formulations. In the case of the lipids we have constructed delivery agents with hyaluronic acid for targeting cancer cells exhibiting CD44 receptors (in collaboration with Esta Sterneck, CCR). Initial experiments look quite positive. In addition, we have working with Jonathan Lovell (U of Buffalo) on the development of photoactivatable polymers for the delivery of our RNA-based nanoparticles. Initial results are also quite encouraging. An invited book on protocols for RNA Nanobiology is now in press, and in addition, several invited review papers and book chapters were also written on the above described subjects.