Recognition of Chromosomal DNA by Chemically Modified Oligonucleotides

描述（由申请人提供）：识别染色体DNA内特定序列的合成剂是控制基因表达的一种很有前途的策略。在之前的资助期间，我们描述了双工rna、肽核酸（PNAs）、rna -肽和锁定核酸（LNAs），它们靶向基因启动子并调节表达。LNA碱基含有一个修饰的核糖，在2'-氧和4'-碳之间有一个亚甲基连接，每次取代可使熔融温度值（Tm）提高100℃。我们将重点放在LNAs上，因为LNAs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LNAs作为研究工具和临床开发的潜力。研究设计。我们将研究agLNAs的机制、优化和应用。在Aim 1中，我们将研究agna介导的核酸靶点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，并为合成低聚物如何影响基因启动子的细胞过程提供重要的基本见解。在目标2中，我们将描述细胞摄取，改进递送方法，并测试化学修饰的LNAs。本Aim的目标是确定最直接和有效的递送方案和化学结构的组合，以实现抗原寡核苷酸的最佳抑制。这一信息将有助于希望使用agLNAs的研究人员，并帮助指导agLNAs用于治疗应用的设计。在Aim 3中，我们提出了使用agLNAs来控制细胞过程的新策略。应用包括使用agLNAs探测染色体DNA内序列的可及性，将agLNAs靶向具有双向启动子的基因（占人类基因的10%），使用agLNAs操纵重要生物医学靶点基因的表达，以及合成和测试aglna -肽偶联物，旨在将转录因子招募到基因启动子并激活基因表达。选择这些实验是因为它们有可能扩大agLNAs的应用范围。生物医学意义。目前对染色体DNA序列特异性识别的选择很少。agLNAs解锁了进入染色体DNA的途径，并为解决在体内获得足够效力和特异性的难题提供了另一种选择。agLNAs还将为探测特定序列的染色体可达性提供有用的工具，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的药物有可能影响与疾病有关的蛋白质的产生，并可能提供一类新的药物。本提案的目标是了解如何识别DNA可以实现，并利用这些知识来制定策略来控制细胞的功能。

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.