Light-activated oligonucleotides for biological applications

描述(由申请人提供)：对大脑活动的遗传基础的分子理解将不可避免地改善与无数神经疾病和障碍相关的医疗干预。剥离大脑分子内部工作的关键一步将是分析大脑转录组，即单个细胞内的信使RNA补体，它控制着多个组织水平的功能-细胞、突触、组织和大脑区域。目前从形态复杂的神经元组织细胞中获取mRNA的方法都是侵入性的，而且容易受到污染。这项建议的生物学目标是在活体小鼠脑片模型中分类不同海马区神经元转录组可变性的差异，然后通过使用条件CREB KO和CREB Ser133Ala敲门小鼠来评估转录组变化的时间和空间方面的响应遗传扰动。CREB是一种转录因子，调节各种cAMP协调行为，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第一次评估CREB在体内的基因表达靶点，其中微环境可能是CREB靶点选择的重要因素。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，它显示了从单个细胞、培养的神经元和大鼠海马脑片中获取mRNA的极有希望。TIVA-TAG是“笼式”寡核苷酸，具有一条PolyU捕获链，在光激活时结合细胞mRNA的PolyA尾巴。这些研究推动了TIVA标签的开发，使其具有更强的体内功能。我们建议优化TIVA标签设计中的许多特征(目标1)，以及开发适合于双光子激活的笼式Ru光敏剂(Ru)TIVA标签(目标2)。最后，我们将通过调整Ru-TIVA上的配体和开发Poly-TIVA，一种互补的笼化技术，使用纳米级、细胞渗透性、近红外响应的聚合体小泡来运送聚-U捕获链(和其他寡核苷酸)，从而开发用于深层组织mRNA采集的多路复用策略(目标3)。RU-TIVA变体和PolyTIVA将扩展同时从组织内多个单个细胞或多个时间点的单个细胞捕获mRNA的能力(即，纵向研究)。这些串联技术应该使评估和潜在地重新编程大脑中的细胞功能成为可能。

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.