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。功能性基因组研究通常已经利用了同源重组，RNA干扰，mRNA/cDNA过表达或其他生物学方法，但是随着我们努力了解体内系统更复杂的过程，这些技术越来越限制。例如，将这些方法应用于特定细胞群体受到我们对顺式调节元件的新知识的阻碍，并且它们可能笨拙地靶向基因组合。它们的动力学需求（例如，CRE重组酶表达的速率，基因组编辑，RNA降解和蛋白质消耗）也降低了可以应用它们的时间精度。 光门控技术可以通过在几秒钟内将多个基因的光学靶向在特定组织中的光学靶向来解决这些局限性。因此，我们的实验室和其他研究小组已经制定了几种笼罩了形态寡核苷酸（MOS）的策略，这些策略是基于在海岸上，海胆，斑马鱼，青蛙和其他产生近代的动物的大量使用这些合成反义试剂的基础。当前的笼式MOS（CMOS）包括发夹，环状，复式或核碱基修饰的探针，但这些技术中的每一种都有缺点：（1）发夹和双链试剂利用抑制性寡核苷酸，这些试剂可以增加其细胞毒性； （2）发夹，循环和双工试剂具有不同程度的“泄漏”； （3）需要多个笼中的核壳以完全阻断MO功能，从而限制了光激活效率。 为了克服这些挑战并为MO照相控制开发了一种通用的方法，我们正在开发一种新的CMO，该CMO采用单一或双向构象。这些新颖的结构中的每一个都利用单个可裂解的系绳来实现末端到后骨（特定的目标1）或末端对基碱基（特定的目标2）链接，并且所得的寡核苷酸曲率和/或核苷酶函数化将防止RNA结合。然后，接头光解会释放这些约束，以允许有效的MO/RNA杂交。我们将探索MO寡核苷酸和各种接头结构中的不同结合位点，以优化套索的CMO功能，并在体外测定RNA功能和良好表征的斑马鱼模型的指导下进行。我们还将评估不同的笼发色团以进行多波长激活，并建立允许同时或顺序基因敲低的组合（特定的目标3）。然后，我们将使用Lariat CMO来揭示胰腺和十二指肠同型因子1（PDX1）以及运动神经元和胰腺同源蛋白蛋白蛋白酶因子1（MNX1）如何合作调节内分泌胰腺发育。这些研究将实验室的专业知识整合在验光探针和斑马鱼模型中，而所得技术将提高我们对分子和系统水平的体内生物学的理解。