Oligonucleotides for turning genes on and off with light

描述（由申请人提供）：提议的光激活寡核苷酸将使以非常高的时空分辨率在活细胞和斑马鱼中“关闭”或“打开”基因成为可能，并使用最先进的紫外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Aim 1中，我们将开发两种互补的方法，通过光来下调基因表达，使用笼化磷酸化DNA （S- DNA）募集RNase H或笼化肽核酸（PNA）立体阻断核糖体。反义（18-25-mer） S-DNA和PNA链将通过光活性连接物结合到长度（8-16 nt）和组成（S-DNA, 2'-OMe RNA， PNA， LNA）可变的互补寡核苷酸上。这些偶联物将通过体外实验进行优化，以实现至少10倍的蛋白下调。在第二阶段，相关的RNA“绷带”将通过光可切割的连接物连接两条短的（6-12mer） 2'-OMe RNA链。将RNA绷带与目标mRNA序列结合将阻断核糖体翻译，直到发生光裂解。RNA绷带相对于单个2'-OMe RNA链的热力学稳定性将通过体外实验进行优化，以实现10倍的蛋白质上调。在Aim 3中，将开发荧光报告器，允许实时监测活标本中的寡核苷酸光激活。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chordin）和神经元（针对GluR2）中进行测试和优化。另外几个基因，包括绿色荧光蛋白，将被上调，作为这些生物系统的概念证明。该项目将通过创造工具来探测癌细胞、斑马鱼和神经元中重要蛋白质的功能，从而改善人类健康。可能的临床应用包括光激活、毒性较低的人类白血病基因疗法。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。我们建议在高空间和时间分辨率下，利用紫外光开发光激活寡核苷酸，用于下调和上调基因表达。这些结构将在血细胞、斑马鱼胚胎和神经元中进行测试和优化。

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.