Fluorescent Nucleosides and Oligonucleotides

项目总结 该计划的目标是设计和合成新的发射核苷和核苷酸。 类似物，并将其实现为用于监测基于核苷和核苷酸的转化的探针，如 以及核酸的功能、结构、动力学和识别。推进基于有效荧光的研究 探索核苷、核苷酸和寡核苷酸及其代谢、调节的工具 与潜在治疗剂的过程和相互作用将进一步了解并推动新的 诊断方法，促进药物发现。具体来说，我们将解决以下问题： 目的1.设计、合成并掺入新的同象荧光核苷和 核苷酸。主要设计标准包括：(I)与天然碱基结构高度相似，以 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吸收的重叠；以及(3)充分的发射 量子效率和可见光发射波长。高效的合成和酶途径将是 设计，提供核苷、核苷酸和寡核苷酸。 目的2.光物理和生物物理表征修饰的核苷和 寡核苷酸。光物理特性(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 通过天然核苷猝灭)将得到严格的评估和解释。这些分析的结果 所产生的数据决定了核苷替代物的用途和潜在应用。 目的3.在生物物理、生化和发现方面实现有前景的发射类似物 化验。这些分析将有助于：(I)研究腺苷的酶脱氨为肌苷，这是 发生在三个关键的生物学背景下：嘌呤代谢、信使核糖核酸编辑和tRNA成熟； C-di-NMPs等发光第二信使及其酶促合成的研究 与核糖开关和蛋白质的相互作用(例如，刺痛)，(Iii)监测RNA-蛋白质结合(例如，k- TURN/7LAE)和翻译事件，包括程序性核糖体移码，这可能是有害的 到天然蛋白质合成，但是，当编程时(例如，在病毒复制中)可以最大限度地表达蛋白质。 核酸在细胞活动中扮演着中心角色，因此对人类免疫缺陷病毒的出现具有巨大的影响。 疾病，进而对人类健康造成危害。这就需要开发新的有效的研究工具 它们的识别特性和外源物质的改造。设计了发射核苷类似物 并将在新的基于实时荧光的分析中实现。这些调查将 促进对与疾病发展和意志有关的关键生物学过程的基本理解 通过促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.