Recognition of Chromosomal DNA by Chemically Modified Oligonucleotides

描述（由申请人提供）：识别染色体DNA内特定序列的合成试剂是控制基因表达的有希望的策略。在上一个资助期间，我们描述了靶向基因启动子和调节表达的双链RNA、肽核酸（PNA）、PNA-肽和锁核酸（LNA）。LNA碱基含有修饰的核糖，其在2 '-氧和4'-碳之间具有亚甲基键，并且每次取代使解链温度值（Tm）增加多达100 ° C。我们把这个建议集中在LNA上，因为LNA是直接识别染色体DNA的最简单和最有效的工具。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agLNAs的机制，优化和应用。在目的1中，我们将研究agLNA介导的核酸靶识别和随后抑制细胞内基因表达的机制。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agLNAs的新应用，并提供重要的基本见解如何合成寡聚体影响基因启动子的细胞过程。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。该目的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，以实现用抗原寡核苷酸的最佳抑制。这些信息将有助于希望使用agLNAs的研究人员，并帮助指导agLNAs的治疗应用设计。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操纵作为重要生物医学靶标的基因的表达，以及合成和测试设计用于将转录因子募集到基因启动子并激活基因表达的agLNA-肽缀合物。选择这些实验是因为它们有可能扩大agLNA的应用范围。生物医学相关性。目前，染色体DNA的序列特异性识别的选择很少。agLNAs开启了对染色体DNA的访问，并为解决在体内实现足够的效力和特异性的难题提供了替代方案。agLNAs还将提供有用的工具，用于探测特定序列处的染色体可及性，这些信息可能导致更好地理解复制、DNA修复和基因表达。公共卫生相关性：染色体DNA编码表达蛋白质所必需的信息。靶向DNA的药物有可能影响与疾病有关的蛋白质的产生，并可能提供一类新的药物。该计划的目标是了解如何识别DNA，并利用这些知识来开发控制细胞功能的策略。

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.