Development of lariat-shaped caged morpholinos for optochemical gene regulation

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、空间和时间特异性的控制基因功能的能力。功能基因组学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体受到我们对顺式调节元件的新生知识的阻碍，并且它们对于靶向基因组合可能是笨拙的。它们的动力学要求（例如，Cre重组酶表达、基因组编辑、RNA降解和蛋白质消耗的速率）也降低了它们可以应用的时间精度。 光选通技术可以通过允许在几秒钟内在特定组织中光学靶向多个基因来解决这些限制。因此，我们的实验室和其他研究小组已经设计了几种策略来笼吗啉代寡核苷酸（MO），建立在这些合成的反义试剂在海鞘，海胆，斑马鱼，青蛙和其他动物，开发子宫外的广泛使用。目前的笼状MO（cMO）包括发夹、环状、双链体或核碱基修饰的探针，但这些技术中的每一种都具有缺点：（1）发夹和双链体试剂利用可增加其细胞毒性的抑制性寡核苷酸;（2）发夹、环状和双链体试剂具有不同程度的“泄漏”;和（3）需要多个笼状核碱基来完全阻断MO功能，限制了光活化效率。 为了克服这些挑战并开发用于MO光控制的通用方法，我们正在开发一类采用单或双构象的新型cMO。这些新结构中的每一种都利用单个光可切割的系链来实现末端至主链（特异性目标1）或末端至碱基（特异性目标2）的连接，并且所得的寡核苷酸弯曲和/或核碱基官能化将阻止RNA结合。然后，接头光解将释放这些约束，以允许有效的MO/RNA杂交。我们将探索MO寡核苷酸内的不同缀合位点和各种接头结构，以优化lymphocMO功能，由RNA功能的体外测定和充分表征的斑马鱼模型指导。我们还将评估不同的笼状发色团的多波长激活，并建立允许同时或顺序基因敲除的组合（具体目标3）。然后，我们将使用lymphocMO来揭示胰腺和十二指肠同源盒因子1（pdx 1）和运动神经元和胰腺同源盒因子1（mnx 1）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，所产生的技术将促进我们在分子和系统水平上对体内生物学的理解。

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.