Oligonucleotides for turning genes on and off with light

用光打开和关闭基因的寡核苷酸

• 批准号: 8214546
• 负责人: Ivan Julian Dmochowski
• 金额: $ 30.41万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-08 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8214546
• 关键词: Antisense Oligonucleotides; Bandage; Blood Cells; Cells; DNA; Dendrites; Development; Down-Regulation; Embryo; Gene Expression; Genes; Health; Human; Individual; Length; Life; Light; MYB gene; Memory; Messenger RNA; Methods; Microscope; Monitor; Names; Neurons; Oligonucleotides; Peptide Nucleic Acids; Principal Investigator; Proteins; Proto-Oncogene Proteins c-myb; RNA; RNA Binding; RNA Stability; Recruitment Activity; Relative (related person); Reporter; Research; Resolution; Ribonuclease H; Ribosomes; Specimen; Testing; Thermodynamics; Time; Translations; Ultraviolet Rays; Up-Regulation; Zebrafish; biological systems; cancer cell; chordin; clinical application; gene therapy; hindbrain; improved; in vitro Assay; in vivo; leukemia; photoactivation; programs; research study; spatiotemporal; tool

DESCRIPTION (provided by applicant): The proposed light-activated oligonucleotides will make it possible to turn genes "off" or "on" in living cells and zebrafish with very high spatiotemporal resolution, and probe the precise functions of different proteins using a state-of-the-art UV confocal microscope. This will build on successful in vivo experiments in the PI's lab using light-activated antisense oligonucleotides. In Aim 1, we will develop two complementary methods for down-regulating gene expression with light using caged phosphorothioated DNA (S- DNA) to recruit RNase H or caged peptide nucleic acid (PNA) to sterically block the ribosome. Antisense (18-25-mer) S-DNA and PNA strands will be conjugated to a complementary oligonucleotide of variable length (8-16 nt) and composition (S-DNA, 2'-OMe RNA, PNA, LNA) via a photoactive linker. These conjugates will be optimized with in vitro assays to achieve at least 10-fold down-regulation of protein. In Aim 2, related RNA "bandages" will be developed by attaching two short (6-12mer) 2'-OMe RNA strands via a photocleavable linker. Binding the RNA bandage to a target mRNA sequence will block ribosomal translation until photocleavage occurs. The thermodynamic stability of the RNA bandages relative to the individual 2'-OMe RNA strands will be optimized using in vitro assays to achieve a 10-fold upregulation of protein. In Aim 3, fluorescent reporters will be developed that allow real-time monitoring of oligonucleotide photoactivation in living specimens. Strategies for down-regulating gene expression will be tested and optimized in leukemia cells (against c-myb), zebrafish (against chordin), and neurons (against GluR2). Several additional genes, including GFP, will be upregulated as proof-of-concept in these biological systems. This project will improve human health by creating tools for probing the function of important proteins in cancer cells, zebrafish, and neurons. Possible clinical applications include light-activated, less toxic gene therapies for human leukemia. Additional studies will explore how the spatial control of protein translation in zebrafish is important in hindbrain development and in dendrites is important for memory formation. We propose to develop light-activated oligonucleotides for down- and up-regulating gene expression with ultraviolet light, at high spatial and temporal resolution. These constructs will be tested and optimized in blood cells, zebrafish embryos, and neurons.

描述（由申请人提供）：所提出的光激活寡核苷酸将使得有可能以非常高的时空分辨率在活细胞和斑马鱼中“关闭”或“打开”基因，并使用最先进的UV共聚焦显微镜探测不同蛋白质的精确功能。这将建立在PI实验室使用光激活反义寡核苷酸的成功体内实验的基础上。在目的1中，我们将开发两种互补的方法，用于用光下调基因表达，使用笼状硫代磷酸化DNA（S-DNA）来募集RNA酶H或笼状肽核酸（PNA）来空间阻断核糖体。反义（18-25-mer）S-DNA和PNA链将通过光敏接头与可变长度（8-16 nt）和组成（S-DNA、2 '-OMe RNA、PNA、LNA）的互补寡核苷酸缀合。这些缀合物将用体外测定法优化以实现蛋白质的至少10倍下调。在目标2中，将通过经由光可切割接头连接两条短（6- 12聚体）2 '-OMe RNA链来开发相关的RNA“绷带”。将RNA绷带与靶mRNA序列结合将阻断核糖体翻译，直到发生光切割。将使用体外测定优化RNA条带相对于单个2 '-OMe RNA链的热力学稳定性，以实现蛋白质的10倍上调。在目标3中，将开发荧光报告，允许实时监测活标本中的寡核苷酸光活化。下调基因表达的策略将在白血病细胞（针对c-myb）、斑马鱼（针对脊索蛋白）和神经元（针对GluR 2）中进行测试和优化。在这些生物系统中，包括GFP在内的几个额外的基因将被上调作为概念验证。该项目将通过创建用于探测癌细胞、斑马鱼和神经元中重要蛋白质功能的工具来改善人类健康。可能的临床应用包括用于人类白血病的光激活、毒性较小的基因疗法。进一步的研究将探讨斑马鱼蛋白质翻译的空间控制在后脑发育中的重要性，以及树突对记忆形成的重要性。我们建议开发光激活寡核苷酸，用于在高空间和时间分辨率下用紫外光下调和上调基因表达。这些构建体将在血细胞、斑马鱼胚胎和神经元中进行测试和优化。