Recognition of Chromosomal DNA by Chemically Modified Oligonucleotides

描述(由申请人提供)：识别染色体DNA中特定序列的合成试剂是控制基因表达的一种有前途的策略。在之前的资助期间，我们描述了以基因启动子为靶标并调节表达的双链RNA、肽核酸(PNA)、PNA-肽和锁定核酸(LNAs)。LNA碱基含有2‘-氧和4’-碳之间的亚甲基连接的改性核糖，每次取代可使熔化温度值(Tm‘s)提高多达100摄氏度。我们将这一建议集中在LNAs上，因为LNAs已经成为直接识别染色体DNA的最简单和最有效的工具。目标。我们在下一个资助期的目标是了解以染色体为靶标的LNA作为研究工具和临床开发指导的潜力。研究设计。我们将对agLNAs的作用机制、优化和应用进行研究。在目标1中，我们将研究agLNA介导的核酸靶标识别以及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转录本、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，并为合成寡聚体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，我们将表征细胞摄取，改进递送方法，并测试化学修饰的LNAs。这一目标的目的是确定最直接和最有效的递送方案和化学结构的组合，以实现对抗基因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，并有助于指导agLNAs用于治疗应用的设计。在目标3中，我们提出了使用agLNAs控制细胞过程的新策略。应用包括利用agLNAs探测染色体DNA中序列的可及性，利用双向启动子将agLNAs靶向基因(占人类基因的10%)，使用agLNAs操纵作为重要生物医学靶点的基因的表达，以及合成和测试agLNA-肽结合物，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。生物医学相关性。目前，对染色体DNA进行序列特异性识别的选择很少。AgLNAs解锁了对染色体DNA的访问，并为解决在体内获得足够的效力和特异性这一难题提供了一种替代方案。AgLNAs还将为探测特定序列上的染色体可及性提供有用的工具，这些信息可能有助于更好地理解复制、DNA修复和基因表达。与公共健康相关：染色体DNA编码表达蛋白质所需的信息。以DNA为靶标的药物有可能影响与疾病相关的蛋白质的生产，并可能提供一类新的药物。这项提议的目标是了解如何实现对DNA的识别，并利用这一知识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.