Promoter Specific Hypermethylation Sensors for Early Cancer Detection

用于早期癌症检测的启动子特异性高甲基化传感器

• 批准号: 7994149
• 负责人: INDRANEEL GHOSH
• 金额: $ 15.61万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994149
• 关键词: Affinity; Apoptosis; BRCA1 gene; Binding; Biosensor; CDH1 gene; Cancer Detection; Cancerous; Cell Cycle Regulation; Chemicals; Clinical; CpG Islands; CpG dinucleotide; Cytosine; DNA; DNA Methyltransferase; DNA Modification Methylases; DNA-Binding Proteins; Detection; Development; Down-Regulation; Early Diagnosis; Ensure; Epigenetic Process; Event; Fingers; Firefly Luciferases; GSTP1 gene; Genes; Genetic Transcription; Genomics; Goals; Green Fluorescent Proteins; Horseradish Peroxidase; Human Genome; Hypermethylation; Incidence; Lactamase; Lead; Luciferases; MGMT gene; Malignant Neoplasms; Methods; Methylation; Modeling; Modification; Morphologic artifacts; Normal tissue morphology; Pattern; Promoter Regions; Proteins; Reporter; Reporting; Research Project Grants; Sampling; Screening for cancer; Series; Site; Specificity; System; Techniques; Technology; Testing; Time; Tissue Sample; Tissues; Tumor Cell Invasion; Tumor Suppressor Genes; Tumor Suppressor Proteins; Zinc Fingers; base; bisulfite; design; ds-DNA; gene repression; human tissue; intercellular communication; maspin; promoter; public health relevance; rapid technique; research study; sensor

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% 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%) 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, 2- Lactamase, and firefly luciferase). Promoter specific hypermethylation will be detected by methyl-CpG binding domains (MBDs), while the correct promoter sequence will recognized by designed zinc-fingers. Our approach has the potential to provide sensitive turn-on sensors 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. PUBLIC HEALTH RELEVANCE: Promoter specific hypermethylation will be detected by methyl-CpG binding domains (MBDs), while the correct promoter sequence will recognized by designed zinc-fingers. Our approach has the potential to provide sensitive turn-on sensors 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.

描述（由申请人提供）： 拟议的研究项目的长期目标是为与癌症有关的特定基因的启动子区域中的CpG岛甲基化直接检测提供一种强大，敏感和快速的方法。胞嘧啶甲基化发生在70-80％的人类基因组的CpG二核苷酸上，最常见于重复的基因组区域。另一方面，CPG岛（定义为具有统计上CpG含量的短序列），其中许多基因的启动子区域（60％）主要受到正常组织中甲基化的影响。这些CpG岛已被发现在癌症中被甲基化，导致转录抑制。最近的实验在众多基因的启动子位点和癌症发生率之间提供了CPG高甲基化之间的密切相关性，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志物。当前检测特定CpG岛甲基化的方法取决于甲基化DNA的广泛的亚硫酸甲基化处理，然后是基于PCR的扩增，测序或微阵列技术。这些当前的方法虽然强大，但也耗时，耗时且昂贵，以表征已知的高甲基化地点。为了快速确定启动子CpG高甲基化，我们将应用我们新开发的技术称为序列启用蛋白质的序列重新组装（SEER）。 SEER系统允许识别双链DNA的特定序列，从而导致功能蛋白报道器（绿色荧光蛋白，2-乳糖酶和萤火虫荧光素酶）的伴随组装。通过甲基-CPG结合结构域（MBD）检测启动子特异性高甲基化，而正确的启动子序列将由设计的锌囊识别。我们的方法有可能提供敏感的转交传感器，以直接报告已知启动子位点的CPG甲基化。如果成功的话，这种方法将在临床环境中迅速区分正常组织和癌组织，而无需用亚硫酸盐处理，PCR扩增和测序。我们将通过1）设计和优化开通生物传感器，以检测模型DNA构建体中的特定甲基化事件； 2）设计和测试生物传感器，以涉及癌症的基因启动子区域（BRCA1，CDH1，P15，P16，MGMT，GSTP1）。 公共卫生相关性： 通过甲基-CPG结合结构域（MBD）检测启动子特异性高甲基化，而正确的启动子序列将由设计的锌囊识别。我们的方法有可能提供敏感的转交传感器，以直接报告已知启动子位点的CPG甲基化。如果成功的话，这种方法将在临床环境中迅速区分正常组织和癌组织，而无需用亚硫酸盐处理，PCR扩增和测序。

项目成果

Direct DNA methylation profiling using methyl binding domain proteins.

• DOI: 10.1021/ac1010316
• 发表时间: 2010-06-15
• 期刊: ANALYTICAL CHEMISTRY
• 影响因子: 7.4
• 作者: Yu, Yinni;Blair, Steve;Gillespie, David;Jensen, Randy;Myszka, David;Badran, Ahmed H.;Ghosh, Indraneel;Chagovetz, Alexander
• 通讯作者: Chagovetz, Alexander