Development of lariat-shaped caged morpholinos for optochemical gene regulation

用于光化学基因调控的套索形笼状吗啉的开发

• 批准号: 9110285
• 负责人: JAMES K CHEN
• 金额: $ 42.48万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9110285
• 关键词: Address; Adopted; Animal Model; Animals; Antisense RNA; Base Pairing; Biological; Biological Assay; Biology; Biomedical Research; Bypass; Cells; Cleaved cell; Complementary DNA; Complex; Development; Disease; Embryo; Evaluation; Gene Activation; Gene Combinations; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Programming; Genomics; Government; Health; Homeobox; In Vitro; Investigation; Kinetics; Knowledge; Laboratories; Light; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Motor Neurons; Neurophysiology - biologic function; Nucleic Acids; Nucleosides; Oligonucleotides; Optics; Organism; Oxygen; Pancreas; Persons; Photoreceptors; Physiology; Phytochrome; Population; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA Degradation; RNA Interference; RNA Splicing; Rana; Reagent; Regulatory Element; Research; Resistance; Resolution; Sea Urchins; Shapes; Site; Specificity; Staging; Structure; System; Technology; Time; Tissues; Transcend; Vertebral column; Whole Organism; Work; Zebrafish; ascidian; base; caged molecule; cell fate specification; chromophore; combinatorial; complex biological systems; crosslink; cryptochrome; cytotoxicity; design; endocrine pancreas development; experience; functional genomics; gene function; genetic inhibitor; genome editing; homologous recombination; in vitro Assay; in vitro activity; in vivo; insulin promoter factor 1; knock-down; light gated; molecular dynamics; mutant; novel; nucleobase; optogenetics; overexpression; photoactivation; photolysis; prevent; recombinase; reverse genetics; spatiotemporal; tool; transcription factor; voltage; zebrafish development

DESCRIPTION (provided by applicant): Deconstructing the molecular basis of normal physiology and disease requires an ability to control gene function with genomic, spatial, and temporal specificity. Functional genomic studies have typically utilized homologous recombination, RNA interference, mRNA/cDNA overexpression, or other biological methods, yet these technologies are increasingly limiting as we strive to understand more complex in vivo systems. For example, applying these methods to specific cell populations is hindered by our nascent knowledge of cis- regulatory elements, and they can be unwieldy for targeting combinations of genes. Their kinetic requirements (e.g., rates of Cre recombinase expression, genome editing, RNA degradation, and protein depletion) also diminish the temporal precision with which they can be applied. Light-gated technologies can address these limitations by allowing the optical targeting of multiple genes in specific tissues within seconds. Accordingly, our laboratories and other research groups have devised several strategies for caging morpholino oligonucleotides (MOs), building upon the extensive use of these synthetic antisense reagents in ascidians, sea urchins, zebrafish, frogs, and other animals that develop ex utero. Current caged MOs (cMOs) include hairpin, cyclic, duplex, or nucleobase-modified probes, yet each of these technologies has drawbacks: (1) hairpin and duplex reagents utilize inhibitory oligonucleotides that can increase their cytotoxicity; (2) hairpin, cyclic, and duplex reagents have varying degrees of "leakiness"; and (3) multiple caged nucleobases are required to completely block MO function, limiting photoactivation efficiency. To overcome these challenges and develop a universal approach for MO photo control, we are developing a new class of cMOs that adopt single- or double-lariat conformations. Each of these novel structures utilizes a single light-cleavable tether to achieve a terminus-to-backbone (Specific Aim 1) or terminus-to-base (Specific Aim 2) linkage, and the resulting oligonucleotide curvature and/or nucleobase functionalization will prevent RNA binding. Linker photolysis will then release these constraints to allow efficient MO/RNA hybridization. We will explore different conjugation sites within the MO oligonucleotide and various linker structures to optimize lariat cMO function, guided by in vitro assays of RNA function and well- characterized zebrafish models. We will also evaluate different caging chromophores for multi-wavelength activation and establish combinations that allow simultaneous or sequential gene knockdowns (Specific Aim 3). We will then use lariat cMOs to uncover how pancreatic and duodenal homeobox factor 1 (pdx1) and motor neuron and pancreas homeobox factor 1 (mnx1) cooperatively regulate endocrine pancreas development. These studies integrate our laboratories' expertise in optochemical probes and zebrafish models, and the resulting technologies will advance our understanding of in vivo biology at the molecular and systems levels.

描述（由申请人提供）：解构正常生理学和疾病的分子基础需要具有基因组、空间和时间特异性控制基因功能的能力。功能基因组研究通常利用同源重组、RNA 干扰、mRNA/cDNA 过表达或其他生物学方法，但随着我们努力了解更复杂的体内系统，这些技术越来越受到限制。例如，将这些方法应用于特定的细胞群受到我们对顺式调控元件的初步了解的阻碍，并且它们对于靶向基因组合来说可能很笨拙。它们的动力学要求（例如 Cre 重组酶表达速率、基因组编辑、RNA 降解和蛋白质消耗）也降低了它们应用的时间精度。 光门控技术可以在几秒钟内光学靶向特定组织中的多个基因，从而解决这些限制。因此，我们的实验室和其他研究小组在海鞘、海胆、斑马鱼、青蛙和其他体外发育动物中广泛使用这些合成反义试剂的基础上，设计了几种笼养吗啉寡核苷酸 (MO) 的策略。目前的笼状 MO (cMO) 包括发夹探针、环状探针、双链体探针或核碱基修饰探针，但这些技术均存在缺点：(1) 发夹探针和双链体试剂使用抑制性寡核苷酸，会增加其细胞毒性； （2）发夹型、环状型、双链体试剂均存在不同程度的“泄漏”； (3)需要多个笼状核碱基来完全阻断MO功能，限制光活化效率。 为了克服这些挑战并开发一种通用的 MO 光控制方法，我们正在开发一类采用单套索或双套索构象的新型 cMO。这些新颖的结构中的每一个都利用单个光可裂解的系链来实现末端到主链（具体目标1）或末端到碱基（具体目标2）的连接，并且所得的寡核苷酸曲率和/或核碱基功能化将阻止RNA 结合。然后连接子光解将释放这些限制，以实现有效的 MO/RNA 杂交。我们将在 RNA 功能体外测定和充分表征的斑马鱼模型的指导下，探索 MO 寡核苷酸内的不同缀合位点和各种接头结构，以优化套索 cMO 功能。我们还将评估不同的笼养生色团的多波长激活，并建立允许同时或顺序基因敲低的组合（具体目标 3）。然后，我们将使用套索 cMO 来揭示胰腺和十二指肠同源盒因子 1 (pdx1) 以及运动神经元和胰腺同源盒因子 1 (mnx1) 如何协同调节内分泌胰腺发育。这些研究整合了我们实验室在光化学探针和斑马鱼模型方面的专业知识，由此产生的技术将增进我们对分子和系统水平的体内生物学的理解。