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

发现和开发人工核酸配体以探测细胞相互作用

• 批准号: 10545036
• 负责人: Prabodhika Mallikaratchy
• 金额: $ 39.13万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10545036
• 关键词: Address; Affinity; Binding; Biology; Buffers; Cell Surface Receptors; Cell surface; Cells; Characteristics; Chemicals; Chemistry; Clinical; Communities; Complex; Decision Making; Development; Engineering; Event; Evolution; Goals; In Vitro; Intervention; Libraries; Ligand Binding; Ligands; Measurement; Mediating; Membrane Proteins; Molecular; Molecular Conformation; Monoclonal Antibodies; Nature; Nucleic Acids; Outcome; Process; RNA; Receptor Cell; Regulation; Sampling; Single-Stranded DNA; Solubility; Specificity; System; T-Lymphocyte; Technology; Work; aptamer; aqueous; base; combinatorial; crosslink; design; innovation; insight; intercellular communication; interest; nanomaterials; receptor; response; scaffold; screening; tool

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.

用于探测细胞相互作用的人工核酸配体的发现和发展 细胞事件的调节，如细胞间通讯，受体- 配体相互作用和受体间的相互作用，使用分子工具，将扩大我们的理解， 由细胞表面受体介导的细胞决策。由于它们的合成性质和相容性 对于多种纳米材料，核酸适体非常适合作为特异性识别探针， 加入这些工具。适体是单链DNA/RNA/XNA（X=非标准核酸 碱基）序列，其以高亲和力和特异性结合至特定靶。然而，由于其低 由于纯化的细胞表面受体在水介质中的溶解度低，因此不能成为体外筛选 适体配体。此外，响应于配体结合，细胞表面受体协同作用，形成 瞬时复合物这样的复合物难以在人工缓冲系统中构成，同时保持其稳定性。 原生褶皱这意味着常规的适体-配体筛选方法可能不会产生适体 是可以翻译的。为了满足这一需求，Mallikaratchy实验室最近开创了一项名为 配体引导的选择或LIGS以从SELEX（系统进化分析）鉴定功能性适体。 通过指数富集的配体）文库，针对其天然的多结构域细胞表面靶标， 功能状态LIGS使用相同的组合文库，但利用了 选择。例如，已知常规SELEX中的迭代过程被设计为胜过 低亲和力结合剂通过竞争过程，其中高亲和力结合剂通过越来越多的 选择过程。LIGS利用组合文库中弱结合剂和强结合剂之间的这种竞争， 引入更强的、已知的高亲和力第二配体，例如，一种单克隆抗体（mAb）， 目标靶标以胜过并取代高度特异性适体。这些适体可以识别它们的 特异性地靶向培养和临床样品中的受体。基于我们对LIGS的初步工作，第一个目标是 这种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转换 膜蛋白的机制/功能的兴趣，以发现人工配体的基础上适体， 他们第二个目标探索化学干预，以了解适体折叠使用生物正交 方法，如点击化学，以促进邻近介导的分子内交联。此外，本发明还 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， 已经使用LIGS鉴定的适体，明确关注T细胞中TCR-CD 3 ε的调节。一 这些目标的成功结果将导致适体能够探测受体相互作用，沿着 集成的纳米材料增强了适体在生物医学中的应用，同时提供了对 细胞决策中细胞受体生物学的调节。