Fluorescent Nucleosides and Oligonucleotides

描述(申请人提供)：该计划的目标是设计和合成新的荧光核苷类似物，并将它们用作核酸结构、动力学和识别的探针。发展有效的基于荧光的工具来探索核酸及其与配体和潜在治疗剂的相互作用将进一步推动新的 这是一种新的诊断方法，将促进药物发现。本项目的具体目标是：目标1。设计、合成和整合新的同象荧光核苷类似物。主要设计标准包括：(I)与天然碱基的结构高度相似，以忠实地模拟它们的大小和形状，以及杂交和识别特性，(Ii)重新移动吸收光谱，以最大限度地减少与天然碱基的吸收重叠，以及(Iii)足够的发射量子效率和长发射波长(最好在可见光范围内)。将设计有效的合成途径，为自动化和酶促寡核苷酸合成提供核苷和必要的构件。目的2.对修饰后的核苷和寡核苷酸进行光物理和生物物理表征。将严格评估和解释光物理特性(如吸收和发射峰值、量子产额和亮度、激发态寿命以及对环境极性的敏感性和天然核苷的静态和动态猝灭)。目的3.实现有前景的发光类似物在生物物理和发现分析中的应用。这些分析将有助于：(I)发现针对细菌核糖体的新抗生素；(Ii)研究RNA解旋酶，即几乎参与RNA新陈代谢的各个方面的马达蛋白；(Iii)监测程序化的核糖体框架转移，这一过程可能对天然蛋白质合成极其有害，但当编程时(如在病毒中)可以最大限度地提高蛋白质的表达；(Iv)研究RNA脱氨化，这是一个重要的转录后过程，使mRNAs和所产生的蛋白质多样化；它与正常的大脑功能有关，当缺陷时与疾病有关，以及(V)RNAi的研究，这是由短干扰RNA(SiRNA)诱导的调节过程，也是能够改变细胞表型、破译遗传途径和确定新的治疗靶点的强大工具。核酸在细胞活动中发挥核心作用，因此对疾病的出现产生巨大影响，进而对人类健康产生巨大影响。这就需要开发新的有效工具来研究它们的识别特性和外源试剂的改变。设计和制备的发射核苷类似物将在新型实时荧光分析中实现。这些研究将促进对与疾病发展有关的关键生物学过程的基本了解，并将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。 公共卫生相关性：核酸(DNA和RNA)在所有细胞活动中发挥核心作用，因此对疾病的出现产生巨大影响，进而对人类健康产生巨大影响。该计划的目标是设计、合成和实施新的发射核苷类似物，作为核酸结构、动力学和识别的探针。基于实时荧光的新方法用于探索核酸及其与潜在治疗药物的相互作用，将加深对与疾病发展相关的关键生物学过程的基本了解，并将通过改进诊断工具和促进药物发现，对改善人类健康产生长期影响。

DESCRIPTION (provided by applicant): The goal of the proposed program is to design and synthesize new fluorescent nucleoside analogs and implement them as probes for nucleic acids structure, dynamics and recognition. Advancing effective fluorescence-based tools for exploring nucleic acids and their interactions with ligands and potential therapeutic agents will further new diagnostic approaches and will facilitate drug discovery. The specific aims of this project are: AIM 1. To design, synthesize and incorporate new isomorphic fluorescent nucleoside analogs. 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) Re shifted absorption spectrum to minimize overlap with the absorption of the natural bases, and (iii) Adequate emission quantum efficiency and long emission wavelengths (preferably in the visible range). Efficient synthetic pathways will be devised, providing the nucleosides and the necessary building blocks for automated and enzymatic oligonucleotide synthesis. 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. AIM 3. To implement the promising emissive analogs in biophysical and discovery assays. These assays will facilitate: (i) The discovery of new antibiotics targeting the bacterial ribosome (ii) The study of RNA helicases, ubiquitous motor proteins, which are involved in nearly every aspect of RNA metabolism, (iii) The monitoring of programmed ribosomal frameshifting, a processes which could be extremely detrimental to native protein synthesis, but, when programmed (e.g., in viruses) can maximize protein expression, (iv) The study of RNA deamination, an important posttranscriptional process, which diversifies mRNAs and the resultant proteins; it is linked to proper brain function and, when defective, to disease, and (v) The study of RNAi, a regulatory process induced by short interfering RNA (siRNA), which is also a powerful tool capable of altering cellular phenotypes, deciphering genetic pathways and identifying new therapeutic targets. 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. PUBLIC HEALTH RELEVANCE: Nucleic acids (DNA and RNA) play central roles in all cellular events and, as such, have immense impact on the emergence of diseases and, in turn, on human health. The goal of the proposed program is to design, synthesize and implement new emissive nucleoside analogs as probes for nucleic acids structure, dynamics and recognition. Novel real-time fluorescence-based methods for exploring nucleic acids and their interactions with potential therapeutic agents 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 diagnostic tools and facilitating drug discovery.