Direct Detection of Hypermethylation in Cancer

描述(申请人提供)：拟议研究项目的长期目标是提供一种可靠、灵敏和快速的方法，用于直接检测与癌症有关的特定基因启动子区域的CpG岛甲基化。胞嘧啶甲基化发生在CpG二核苷酸处，在人类基因组的70%-80%，最常见的是重复的基因组区域。另一方面，CpG岛存在于许多基因的启动子区域(60%)，在正常组织中主要保护不受甲基化的影响。已发现这些CpG岛在癌症中发生甲基化，导致转录抑制。最近的实验提供了许多基因启动子位置的CpG高甲基化与癌症发病率之间的强烈相关性，从而使特定的启动子高甲基化成为早期检测的有价值的标记。目前检测特定CpG岛甲基化的方法依赖于对甲基化DNA进行广泛的亚硫酸氢盐处理，然后是基于PCR的扩增、测序或微阵列技术。这些目前的方法虽然功能强大，但对于描述已知的高甲基化位点来说，也是费力、耗时和昂贵的。为了实现快速确定启动子CpG超甲基化的目标，我们将应用我们最新开发的称为蛋白质序列启用重组(SEER)的技术。SEER系统允许识别双链DNA的特定序列，从而导致功能蛋白报告器(绿色荧光蛋白和β-内酰胺酶)的伴随组装。在拟议的特定CpG超甲基化检测中，我们将利用Mbd2蛋白的甲基-CpG结合结构域(MBD)来靶向CpG岛，同时利用设计的锌指来靶向正确的启动子序列。我们的方法有可能提供一个灵敏的启动传感器，用于直接报告已知启动子位置的CpG甲基化。这种方法，如果成功，将在临床环境中快速区分正常组织和癌症组织，而不需要亚硫酸氢盐治疗、聚合酶链式反应扩增和测序。我们将通过以下方式提供概念证明：1)设计和优化用于检测模型DNA结构中特定甲基化事件的启动型生物传感器；2)设计和测试针对与癌症有关的基因(BRCA1、CDH1、p15、p16、MGMT、GSTP1)启动子区的生物传感器。

DESCRIPTION (provided by applicant): The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80 percent of the human genome, most often in repetitive genomic regions. On the other hand, CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60 percent) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer, leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and beta-lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach, if successful, will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs; and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer.