Fluorescent Nucleosides and Oligonucleotides

项目摘要 该项目的目标是设计和合成新的发射性核苷和核苷酸 类似物，并将它们作为探针用于监测基于核苷和核苷酸的转化， 以及核酸的功能、结构、动力学和识别。推进有效的基于荧光的 研究核苷、核苷酸和寡核苷酸及其代谢、调节 过程和与潜在治疗剂的相互作用将进一步了解和推进新的 诊断方法，促进药物发现。具体而言，我们将解决： AIM 1.设计、合成和掺入新型同晶型荧光核苷， 个核苷酸主要设计标准包括：（i）与天然核碱基的高度结构相似性， 忠实地模仿它们的大小和形状，以及杂交和识别特性，（ii）红移 吸收光谱，以最大限度地减少与天然碱的吸收重叠，以及（iii）适当的排放 量子效率和可见发射波长。有效的合成和酶促途径将是 设计，提供核苷、核苷酸和寡核苷酸。 AIM 2.对修饰的核苷进行免疫学和生物免疫学表征， 寡核苷酸物理特性（例如，吸收和发射最大值，量子产率 和亮度，激发态寿命，以及对环境极性和静态和动态的敏感性 被天然核苷猝灭）将被严格地评估和解释。这些分析的结果 并且产生的数据指示核苷替代物的效用和潜在应用。 AIM 3.在生物物理，生物化学和发现中实现有前途的发射类似物 测定。（i）研究腺苷到肌苷的酶促脱氨作用， 发生在三个关键的生物学背景下：嘌呤代谢，mRNA编辑和tRNA成熟;（ii） 研究发射性第二信使（如c-di-NMP）的酶促合成，以及它们的生物学活性。 与核糖开关和蛋白质的相互作用（例如，（iii）监测RNA-蛋白结合（例如，k- turn/7 LAe）和翻译事件，包括程序性核糖体移码，这可能是有害的 天然蛋白质合成，但是，当编程时（例如，在病毒复制中）可以使蛋白质表达最大化。 核酸在细胞事件中起着核心作用，因此，对细胞的出现有着巨大的影响。 疾病，进而影响人类健康。这就需要开发新的有效工具来研究 它们的识别特性和被外源因子改变。所设计的发射性核苷类似物 并将在新的基于真实的实时荧光的测定中实施。这些调查将 进一步了解与疾病发展有关的关键生物学过程， 通过推进知识和促进药物发现，对改善人类健康产生长期影响。

PROJECT SUMMARY The goal of the proposed program is to design and synthesize new emissive nucleoside and nucleotide analogs and implement them as probes for monitoring nucleoside- and nucleotide-based transformations as well as nucleic acids function, structure, dynamics and recognition. Advancing effective fluorescence-based tools for exploring nucleoside, nucleotide and oligonucleotides, as well as their metabolism, regulatory processes and interactions with potential therapeutic agents will further knowledge and advance new diagnostic approaches, facilitating drug discovery. Specifically, we will address: AIM 1. To design, synthesize and incorporate new isomorphic fluorescent nucleoside and nucleotides. The main design criteria include: (i) High structural similarity to the native nucleobases to faithfully mimic their size and shape, as well as hybridization and recognition properties, (ii) Red shifted absorption spectrum to minimize overlap with the absorption of the natural bases, and (iii) Adequate emission quantum efficiency and visible emission wavelengths. Efficient synthetic and enzymatic pathways will be devised, providing the nucleosides, nucleotides and oligonucleotide. AIM 2. To photophysically and biophysically characterize the modified nucleosides and oligonucleotides. The photophysical characteristics (e.g., absorption and emission maxima, quantum yield and brightness, excited state lifetime, as well as susceptibility to environmental polarity and static and dynamic quenching by native nucleosides) will be rigorously evaluated and interpreted. The outcome of these analyses and the data generated dictate the utility and potential applications of the nucleoside surrogates. AIM 3. To implement the promising emissive analogs in biophysical, biochemical and discovery assays. These assays will facilitate: (i) Studying the enzymatic deamination of adenosine to inosine, which occurs in three key biological contexts: purine metabolism, mRNA editing and tRNA maturation; (ii) Investigating the enzymatic synthesis of emissive second messengers, such as c-di-NMPs, and their interactions with riboswitches and proteins (e.g., STING), (iii) Monitoring RNA–protein binding (e.g., k- turn/7LAe) and translational events, including programmed ribosomal frameshifting, which could be detrimental to native protein synthesis, but, when programmed (e.g., in viral replication) can maximize protein expression. Nucleic acids play central roles in cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. This necessitates the development of new effective tools for studying their recognition properties and alteration by exogenous agents. The emissive nucleoside analogs designed and prepared will be implemented in novel real time fluorescence-based assays. These investigations will further the fundamental understanding of key biological processes related to disease development and will have long-term impact on improving human health by advancing knowledge and facilitating drug discovery.