Discovery and development of artificial nucleic acid ligands to probe cellular interactions

用于探测细胞相互作用的人工核酸配体的发现和发展 随后测量细胞事件的调节，如细胞间通信、受体- 配体相互作用，以及使用分子工具的受体间相互作用，将扩大我们对 细胞表面受体介导的细胞决策。由于它们的综合性和兼容性 由于具有多种纳米材料，核酸适配子非常适合作为特异性识别探针 并入这样的工具。适配子是单链DNA/RNA/XNA(X=非标准核酸 碱基)具有高亲和力和特异性地与特定靶标结合的序列。然而，由于他们的低收入 在水介质中的溶解性，纯化的细胞表面受体不是体外筛选的良好靶点 适体配体。此外，作为对配体结合的反应，细胞表面受体协同作用，形成 瞬变复合体。这种络合物很难在人工缓冲系统中组成，同时保持其 原生褶皱。这意味着传统的适配子筛选方法可能不会产生适配子。 是可翻译的。为了满足这一需求，Mallikaratchy实验室最近率先开发了一种名为 从SELEX中识别功能性适配子的配基引导选择或LIGS(系统进化 通过指数浓缩的配体)文库，对照其天然的已知多域细胞表面靶标 功能状态。Ligs使用相同的组合库，但利用了 塞莱克斯。例如，众所周知，常规SELEX中的迭代过程旨在胜过竞争对手 低亲和力粘合剂通过竞争过程使高亲和力粘合剂通过越来越多的 有选择的过程。Ligs利用组合库中弱绑定器和强绑定器之间的竞争，通过 引入一种更强的、已知的高亲和力的二级配基，例如，针对 感兴趣的目标是竞争优势并取代高度特异的适配子。这些适配子可以识别他们的 培养和临床样本中特异的靶向受体。在我们最初关于LLS的工作的基础上，第一个目标是 Mira的目的是将Ligs中使用的相互作用类型扩展到自然诱导的构象开关 机械/功能感兴趣的膜蛋白发现基于核酸适体的人工配体 他们。第二个目标是探索利用生物正交法了解适配子折叠的化学干预。 方法，如点击化学，以促进邻近介导的分子内交联。另外， 我们计划设计功能性适体支架来调节细胞-细胞和受体-配体的相互作用 已经确定了使用LIGS的适体，明确地专注于T细胞中TCR-CD3ε的调节。一个 这些目标的成功结果将导致适体能够探测受体相互作用以及 集成纳米材料增强适体在生物医学中的应用，同时提供对 细胞受体生物学在细胞决策中的调控。

Discovery and development of artificial nucleic acid ligands to probe cellular interactions The regulation followed by measurement of cellular events, such as intercellular communication, receptor- ligand interactions, and inter-receptor interactions using molecular tools, will expand our understanding of cellular decision-making mediated by cell surface receptors. Owing to their synthetic nature and compatibility with a variety of nano-materials, nucleic acid aptamers are well suited as specific recognition probes for incorporation into such tools. Aptamers are single-stranded DNA/RNA/XNA (X= nonstandard nucleic acid base) sequences that bind to a specific target with high affinity and specificity. However, because of their low solubility in aqueous media, purified cell-surface receptors do not make good targets for in vitro screening of aptamer ligands. Additionally, in response to ligand binding, cell surface receptors act in concert, forming transient complexes. Such complexes are hard to constitute in artificial buffer systems while maintaining their native fold. This means that conventional approaches to aptamer-ligand screening may not lead to aptamers that are translatable. Addressing this need, the Mallikaratchy Lab recently pioneered a technology termed Ligand-guided Selection or LIGS to identify functional aptamers from a SELEX (Systematic Evolution of Ligands by EXponential enrichment) library, against known multi-domain cell surface targets in their native functional state. LIGS uses the same combinatorial library but takes advantage of characteristics inherent to SELEX. For example, it is known that the iterative process in conventional SELEX is designed to outcompete low-affinity binders through a competitive process whereby high-affinity binders move through an increasingly selective process. LIGS exploits this competition between weak and strong binders in a combinatorial library by introducing a stronger, known high-affinity secondary ligand, e.g., a monoclonal antibody (mAb), against the target of interest to outcompete and replace highly specific aptamers. These aptamers can recognize their target receptors in cultured and clinical samples specifically. Building on our initial work on LIGS, the first goal of this MIRA is to extend the types of interactions utilized in LIGS to naturally induced conformational switches of membrane proteins of mechanistic/functional interest to discover artificial ligands based on aptamers against them. The second goal explores chemical interventions to understand aptamer folding using bioorthogonal approaches, such as click-chemistry, to facilitate proximity mediated intra-molecular cross-linking. Additionally, we plan to engineer functional aptamer scaffolds to regulate cell-cell and receptor-ligand interactions using aptamers already identified using LIGS, explicitly focusing on the modulation of TCR-CD3ε in T-cells. A successful outcome to these goals will result in aptamers able to probe receptor interactions along with integrated nano-materials enhancing the application of aptamers in biomedicine while providing insights into the modulation of cell receptor biology in cellular decision making.