Recognition of Chromosomal DNA by Chemically Modified Oligonucleotides

化学修饰寡核苷酸对染色体 DNA 的识别

• 批准号: 7770836
• 负责人: David R Corey
• 金额: $ 29.84万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7770836
• 关键词: Achievement; Address; Affect; Androgen Receptor; Antisense Oligonucleotides; Apolipoproteins B; Carbon; Cell physiology; Cells; Cellular biology; Chemical Structure; Chromosomes; Chronic Lymphocytic Leukemia; Clinical; Clinical Treatment; Clinical Trials; Collaborations; Complementary DNA; Complex; Confocal Microscopy; DNA; DNA Repair Gene; Development; Digestion; Disease; Environment; Funding; Future; Gene Expression; Gene Expression Regulation; Gene Structure; Gene Targeting; Genes; Goals; Human; Knowledge; Label; Laboratories; Lead; Learning; Letters; Life; Measures; Mediating; Messenger RNA; Methods; Modeling; Modification; Molecular Target; Nucleic Acids; Oligonucleotides; Oxygen; Peptide Nucleic Acids; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; Production; Progesterone Receptors; Promoter Regions; Proteins; Protocols documentation; RNA; RNA Sequences; Recruitment Activity; Research; Research Design; Research Personnel; Resistance; Ribose; Specificity; Structure-Activity Relationship; Temperature; Testing; Therapeutic; Transcript; antigene; base; carbene; cost; design; experience; fluorophore; improved; in vivo; insight; locked nucleic acid; melting; novel; novel strategies; nuclease; promoter; public health relevance; research study; tool; transcription factor; uptake

DESCRIPTION (provided by applicant): Synthetic agents that recognize specific sequences within chromosomal DNA are a promising strategy for controlling gene expression. During the previous funding period we described duplex RNAs, peptide nucleic acids (PNAs), PNA-peptides, and locked nucleic acids (LNAs) that target gene promoters and modulate expression. LNA bases contain a modified ribose with a methylene linkage between the 2'-oxygen and 4'-carbon and increase melting temperature values (Tm's) by as much as 100C per substitution. We focus this proposal on LNAs because LNAs have been the simplest and most effective tools for direct recognition of chromosomal DNA. Objectives. Our objective for the next funding period is to understand the potential of chromosome- targeted LNAs as research tools and leads for clinical development. Research Design. We will investigate the mechanism, optimization and applications of agLNAs. In Aim 1 we will investigate the mechanism of agLNA-mediated recognition of nucleic acid targets and subsequent inhibition of gene expression inside cells. The environment surrounding gene promoters is a complex mix of RNA transcripts, proteins, and chromosomal DNA. Understanding the mechanism of recognition will help guide use of agLNAs for new applications and provide important basic insights into how synthetic oligomers affect cellular processes at gene promoters. In Aim 2, we will characterize cellular uptake, improve delivery methods, and test chemically modified LNAs. The goal for this Aim is to identify the most straightforward and efficient combination of delivery protocol and chemical structure for achieving optimal inhibition with antigene oligonucleotides. This information will assist researchers hoping to use agLNAs and help guide the design of agLNAs for therapeutic applications. In Aim 3 we propose novel strategies to use agLNAs to control cellular processes. Applications include use of agLNAs to probe accessibility of sequences within chromosomal DNA, targeting agLNAs to genes with bidirectional promoters (10 % of human genes), using agLNAs to manipulate expression of genes that are important biomedical targets, and synthesis and testing of agLNA-peptide conjugates designed to recruit transcription factors to gene promoters and activate gene expression. These experiments were chosen for their potential to expand the range of applications for agLNAs. Biomedical Relevance. There are currently few options for sequence-specific recognition of chromosomal DNA. agLNAs unlock access to chromosomal DNA and provide an alternative for addressing the difficult problem of achieving adequate potency and specificity in vivo. agLNAs will also provide useful tools for probing chromosome accessibility at specific sequences, information that may lead to a better understanding of replication, DNA repair, and gene expression. PUBLIC HEALTH RELEVANCE: Chromosomal DNA encodes the information necessary to express proteins. Agents that target DNA have the potential to affect production of proteins involved in disease and may provide a new class of drugs. The goals of this proposal are to learn how recognition of DNA can be achieved and to use this knowledge to develop strategies for controlling the function of cells.

描述（由申请人提供）：识别染色体 DNA 内特定序列的合成剂是控制基因表达的有前途的策略。在之前的资助期间，我们描述了靶向基因启动子并调节表达的双链体 RNA、肽核酸 (PNA)、PNA 肽和锁定核酸 (LNA)。 LNA 碱基含有修饰核糖，其 2'-氧和 4'-碳之间具有亚甲基键，每次取代可将熔解温度值 (Tm) 提高多达 100°C。我们将此提案的重点放在 LNA 上，因为 LNA 是直接识别染色体 DNA 的最简单、最有效的工具。目标。我们下一个资助期的目标是了解染色体靶向 LNA 作为研究工具和临床开发先导的潜力。研究设计。我们将研究agLNA的机制、优化和应用。在目标 1 中，我们将研究 agLNA 介导的核酸靶标识别以及随后细胞内基因表达抑制的机制。基因启动子周围的环境是 RNA 转录物、蛋白质和染色体 DNA 的复杂混合物。了解识别机制将有助于指导 agLNA 在新应用中的使用，并为合成寡聚物如何影响基因启动子处的细胞过程提供重要的基本见解。在目标 2 中，我们将表征细胞摄取、改进递送方法并测试化学修饰的 LNA。该目标的目标是确定递送方案和化学结构的最直接和有效的组合，以实现反基因寡核苷酸的最佳抑制。这些信息将帮助希望使用 agLNA 的研究人员，并帮助指导用于治疗应用的 agLNA 的设计。在目标 3 中，我们提出了使用 agLNA 控制细胞过程的新策略。应用包括使用 agLNA 探测染色体 DNA 内序列的可及性、将 agLNA 靶向具有双向启动子的基因（10% 的人类基因）、使用 agLNA 操纵重要生物医学靶标基因的表达以及 agLNA 肽的合成和测试旨在招募转录因子到基因启动子并激活基因表达的缀合物。选择这些实验是因为它们具有扩大 agLNA 应用范围的潜力。生物医学相关性。目前用于染色体 DNA 序列特异性识别的选择很少。 agLNA 解锁了染色体 DNA 的获取途径，并为解决体内实现足够效力和特异性的难题提供了一种替代方案。 agLNA 还将提供有用的工具来探测特定序列的染色体可及性，这些信息可能有助于更好地理解复制、DNA 修复和基因表达。公共卫生相关性：染色体 DNA 编码表达蛋白质所需的信息。靶向 DNA 的药物有可能影响疾病相关蛋白质的产生，并可能提供一类新的药物。该提案的目标是了解如何实现 DNA 识别，并利用这些知识来制定控制细胞功能的策略。