Oligonucleotides for turning genes on and off with light

用光打开和关闭基因的寡核苷酸

基本信息

批准号：
7352657
负责人：
Ivan Julian Dmochowski
金额：
$ 29.23万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-02-08 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7352657
关键词：
Antisense DNA Antisense Oligonucleotides Arts Bandage Base Pairing Binding Biochemical Biological Biological Assay Blood Cells Cells Cleaved cell Combinatorial Synthesis DNA DNA Binding Dendrites Development Down-Regulation Embryo Exposure to Fluorescein Fluoresceins Fluorescence Resonance Energy Transfer Gene Expression Genes Genetic Translation Glutamates Green Fluorescent Proteins Health Human In Vitro Individual Initiator Codon K562 Cells Laser Scanning Confocal Microscopy Lasers Length Life Light Link Linker DNA Longevity MYB gene Measures Memory Messenger RNA Methods Microscope Molecular Monitor Neurons Nucleotides Oligonucleotides Oncogenic Oryctolagus cuniculus Peptide Nucleic Acids Protein Biosynthesis Proteins Proto-Oncogene Proteins c-myb RNA RNA Binding RNA Stability Recruitment Activity Relative (related person)Reporter Resolution Reticulocytes Ribonuclease H Ribosomes Role Specimen Standards of Weights and Measures System Testing Thermodynamics Time Transcriptional Activation Translations Ultraviolet Rays Up-Regulation Vertebral column Zebrafish analog base cancer cell chordin clinical application concept cyanine dye 5 design gene repression gene therapy hindbrain improved in vitro Assay in vivo latent nuclear antigen leukemia mRNA Transcript Degradation melting photoactivation prevent protein expression prototype research study spatiotemporal tool translation assay ultraviolet irradiation uptake

项目摘要

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）：提出的光激活寡核苷酸将使活细胞和斑马鱼的时空分辨率在活细胞和斑马鱼中“ OFF”或“ ON”，并探测使用尚未达到的蛋白质的精确功能，并探测不同蛋白质的精确功能。这将建立在PI实验室中成功的体内实验的基础上，使用光激活的反义寡核苷酸。在AIM 1中，我们将开发两种互补方法，用于使用笼子磷酸盐纤维的DNA（S- DNA）在光中下调基因表达，以募集RNase H或笼中的肽核酸（PNA），以阻断核糖体。反义（18-25-mer）S-DNA和PNA链将通过光活动链接器将可变长度（8-16 NT）和组成（S-DNA，2'-OME RNA，PNA，LNA）的互补寡核苷酸偶联。这些结合物将通过体外测定进行优化，以达到至少10倍的蛋白质下调。在AIM 2中，相关的RNA“绷带”将通过连接两个短（6-12mer）2'-Ome RNA链通过光电接头来开发。将RNA绷带与靶mRNA序列结合将阻塞核糖体翻译，直到发生光切除。 RNA绷带相对于单个2'-OME RNA链的热力学稳定性将使用体外测定进行优化，以实现蛋白质的10倍上调。在AIM 3中，将开发荧光记者，以实时监测活样本中的寡核苷酸光活化。将在白血病细胞（针对C-MYB），斑马鱼（针对Chordin）和神经元（针对GLUR2）中测试和优化下调节基因表达的策略。在这些生物系统中，包括GFP在内的其他几个基因将被上调为概念验证。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人类白血病的光激活，毒性较小的基因疗法。其他研究将探讨斑马鱼中蛋白质翻译的空间控制在后脑发育中至关重要，而树突中的蛋白质翻译对于记忆形成至关重要。我们建议在高空间和时间分辨率下开发光激活的寡核苷酸，以紫外线向下和上调基因表达。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。