Light-activated oligonucleotides for biological applications

描述（由申请人提供）：对大脑活动遗传基础的分子理解将不可避免地改善与无数神经系统疾病和障碍相关的医疗干预。揭开大脑分子内部运作的关键一步是分析大脑转录组，即单个细胞内的信使RNA互补物，它控制着组织细胞、突触、组织和大脑区域的多个层次的功能。目前用于从形态复杂的神经元组织中的细胞收获mRNA的方法是侵入性的并且易于污染。 该建议的生物学目标是在活小鼠脑切片模型中对不同海马区域中神经元转录组变异性的差异进行编目，然后通过使用条件性CREB KO和CREB Ser 133 Ala敲入小鼠来评估转录组变化对遗传扰动的响应的时间和空间方面。 CREB是一种转录因子，调节各种cAMP协调的行为，包括学习和记忆，药物成瘾和恐惧条件反射。 我们在这些模型系统中的目标将是首次评估CREB在体内的基因表达靶点，其中微环境可能是CREB靶点选择的重要贡献者。 为了应对这些挑战，我们开发了一种光激活转录组体内分析（TIVA）标签，该标签显示出从培养的神经元和大鼠海马脑切片中的单个细胞中收集mRNA的极端前景。TIVA标签是“笼状”寡核苷酸，具有在光活化时结合细胞mRNA的聚腺苷酸尾的聚腺苷酸捕获链。这些研究激发了具有更大体内功能的TIVA标签的开发。我们提出了TIVA-标签设计（目标1）中的许多功能的优化，以及笼状钌光敏剂（Ru）-TIVA-标签适合于2-光子激活（目标2）的发展。最后，我们将通过调整Ru-TIVA上的配体和开发poly-TIVA（一种用于使用纳米级、细胞可渗透、近红外响应性聚合物囊泡（Aim 3）递送poly-U捕获链（和其他寡核苷酸）的互补笼化技术），开发用于深层组织mRNA收获的多路复用策略。Ru-TIVA变体和多聚TIVA将扩展从组织内的多个单独细胞或在多个时间点从单个细胞同时捕获mRNA的能力（即，纵向研究）。这些串联技术应该可以评估和潜在地重新编程大脑中的细胞功能。

DESCRIPTION (provided by applicant): A molecular understanding of the genetic basis for brain activity will inevitably improve medical interventions relating to myriad neurological conditions and disorders. A critical step in peeling back the molecular inner-workings of the brain will be to assay the brain transcriptome, the messenger RNA complement within individual cells that controls function at multiple levels of organization-cell, synapse, tissue, nd brain region. Current methods for harvesting mRNA from cells in morphologically complex neuronal tissue are invasive and prone to contamination. Biological goals of this proposal are to catalog differences in neuronal transcriptome variability in different hippocampal regions in the live mouse brain slice model, and then to assess temporal and spatial aspects of transcriptome changes in response to genetic perturbation through the use of conditional CREB KO and CREB Ser133Ala knockin mice. CREB is a transcription factor that modulates various cAMP-coordinated behaviors including learning and memory, drug addiction and fear conditioning. Our goal in these model systems will be to assess, for the first time, the gene expression targets of CREB in vivo where the microenvironment will likely be an important contributor to the selection of CREB targets. To address these challenges, we have developed a light-activated transcriptome in vivo analysis (TIVA)-tag that shows extreme promise for harvesting mRNA from individual cells, in cultured neurons and rat hippocampal brain slices. TIVA-tags are "caged" oligonucleotides, with a poly-U capture strand that binds the poly-A tail of cellular mRNA upon photoactivation. These studies motivate the development of TIVA-tags with even greater in vivo functionality. We propose the optimization of many features in the TIVA-tag design (Aim 1), as well as development of caged ruthenium photosensitizer (Ru)-TIVA-tags suitable for 2-photon activation (Aim 2). Finally, we will develop multiplexing strategies for deep-tissue mRNA harvesting by tuning the ligands on Ru-TIVA and by developing poly-TIVA, a complementary caging technology for delivering poly-U capture strands (and other oligonucleotides) using nanoscale, cell-permeable, near-IR-responsive polymersome vesicles (Aim 3). Ru-TIVA variants and poly- TIVA will extend the capability to capture simultaneously mRNA from multiple individual cells within a tissue, or from single cells at multiple time points (i.e., longitudinal studies). These tandem technologies should make it possible to assess and potentially reprogram cellular function in the brain.