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。获得新的表达蛋白质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”具有存活和迁移优势。顺式作用序列、RNA结合蛋白或上游信号传导途径的改变影响编码原癌基因、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，以促进肿瘤发生和癌症进展，特别是在由缺氧或活性氧（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形成，这是翻译抑制和eIF 21磷酸化的结果，其可以抑制细胞凋亡。在本申请中，我们将专注于开发技术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，以及2）通过表征细胞/组织环境中的mRNA/RNA结合蛋白相互作用。为了做到这一点，我们将进一步开发探针来成像固定和活细胞中的低拷贝数天然mRNA，并开发一种新的方法来检测RNA-蛋白质相互作用，具有单一的相互作用灵敏度。最近，我们已经开发了一种新的探针组成的四个高亲和力，耐核酸酶，线性核酸，标记有多个，高量子产率的荧光团连接在一起的链霉亲和素，通过生物素链霉亲和素连接。这种设计产生多价、单一RNA敏感和通用的成像探针。由于这些探针在其核心含有蛋白质，链霉亲和素，这是我们的论点，当结合抗体为基础的邻位连接试验（PLA）和荧光显微镜，单一的RNA-蛋白质相互作用，可以在固定的组织或活细胞杂交后，固定的细胞样品中检测。邻位连接测定法利用寡核苷酸连接的抗体、连接、滚环扩增（RCA）和荧光检测来实现单一相互作用灵敏度。最后，一旦确定了这些相互作用，将为相关的RNA结合蛋白开发荧光蛋白，以允许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（固定和活的）和临床肿瘤样本中表征这些事件。从这一特征，我们希望确定新的目标，为治疗的发展，并可能获得深入了解的机制，可能使肿瘤细胞难治性化疗和放疗。

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.