Characterizing gene regulation with single molecule sensitive probes

描述（由申请人提供）：基因表达的失调导致癌细胞的异常表型和行为。获得表达蛋白的新谱及其随后的激活使癌细胞能够重新进入细胞周期，或使它们比“正常细胞”具有生存和迁移优势。顺式作用序列、RNA结合蛋白或上游信号通路的改变会影响编码原癌基因、细胞因子、细胞周期调节因子和其他调节蛋白的mrna的稳定性和/或翻译效率，从而促进肿瘤的发生和癌症的进展，特别是在缺氧或活性氧（ROS）诱导的细胞应激期间。此外，这些环境还可以诱导应激颗粒的形成，这是翻译抑制和eIF21磷酸化的结果，从而抑制细胞凋亡。在本应用中，我们将专注于开发技术，通过以下方式来表征基因表达的失调：1)通过评估已经转录的mRNA来评估癌细胞的转录活性；2)通过表征细胞/组织环境中mRNA/RNA结合蛋白的相互作用。为此，我们将进一步开发探针，对固定细胞和活细胞中的低拷贝数天然mrna进行成像，并开发一种具有单相互作用灵敏度的检测rna -蛋白相互作用的新方法。最近，我们开发了一种新的探针，由四种高亲和力，耐核酸酶的线性核酸组成，通过生物素-链霉亲和素连锁，用多个高量子产率的荧光团标记在一起。这种设计产生了一种多价、单RNA敏感和通用的成像探针。由于这些探针的核心含有一种蛋白质链亲和素，因此我们认为，当结合基于抗体的近距离连接试验（pla）和多色荧光显微镜时，可以在固定组织或活细胞杂交后的固定细胞样品中检测到单个rna -蛋白质相互作用。邻近连接试验利用寡核苷酸连接抗体，连接，滚环扩增（RCA）和荧光检测，以实现单一相互作用的敏感性。最后，一旦这些相互作用被确定，将开发出相关RNA结合蛋白的荧光蛋白，以便在癌症相关过程中监测活细胞中的这些相互作用。因此，通过这项技术的发展，我们将能够在前列腺肿瘤进展的细胞模型中描述这些事件，无论是固定的还是活的，以及在临床肿瘤样本中。从这一特性中，我们希望确定治疗发展的新靶点，并可能深入了解可能使肿瘤细胞对化疗和放疗难以耐受的机制。

DESCRIPTION (provided by applicant): Deregulation of gene expression contributes to aberrant phenotypes and behaviors of cancer cells. Acquiring a new profile of expressed proteins and their subsequent activation enable cancer cells to re-enter the cell cycle, or give them survival and migratory advantages over those of the "normal cells". Alterations in cis-acting sequences, RNA binding proteins, or in upstream signaling pathways affect the stability and/or translational efficiency of mRNAs encoding proto-oncogenes, cytokines, cell cycle regulators and other regulatory proteins to promote tumorigenesis and cancer progression, especially during cellular stress induced by hypoxia or reactive oxygen species (ROS). In addition, these environments can also induce stress granule formation, a consequence of translational suppression and eIF21 phosphorylation, which can inhibit apoptosis. In this application we will focus on developing technology to characterize the deregulation of gene expression by: 1) evaluating the transcriptional activity of cancer cells by assessing mRNAs already transcribed, and 2) by characterizing mRNA/RNA binding protein interactions in the cellular/tissue milieu. To do this we will further develop probes to image low copy number native mRNAs in both fixed and living cells, and develop a novel method for detecting RNA-protein interactions with single interaction sensitivity. Recently we have developed a new probe consisting of four high-affinity, nuclease resistant, linear nucleic acids, labeled with multiple, high quantum-yield fluorophores linked together by streptavidin, via the biotin-streptavidin linkage. This design results in a multivalent, single RNA sensitive and versatile imaging probe. Because these probes contain a protein at their core, streptavidin, it is our contention that when combined with antibody-based proximity ligation assays (PLAs) and multicolor fluorescence microscopy, single RNA-protein interactions can be detected in fixed tissue or post live-cell hybridization, fixed cell samples. Proximity ligation assays utilize oligonucleotide-linked antibodies, ligation, rolling circle amplification (RCA) and fluorescence detection to achieve single interaction sensitivity. Last, once these interactions are identified, fluorescent proteins will be developed for the relevant RNA binding proteins to allow for the monitoring of these interactions in live cells during cancer relevant processes. Thus, through the development of this technology, we will be able to characterize these events in cellular models of prostate tumor progression, both fixed and live, and in clinical tumor samples. From this characterization we hope to identify new targets for therapeutic development and possibly gain insight into mechanisms that may make tumor cells refractory to chemo and radiation therapy.