METHYL-SENTRY: Proposed feasibility study of a nanopore diagnostic tool with rapid automated measurement of cell free DNA methylation state for clinical cancer evaluation

甲基哨兵：拟议的纳米孔诊断工具的可行性研究 指示癌症状态的游离DNA中的高甲基化生物标志物 癌症的早期发现与改善预后和降低发病率相关，因为更及时的 治疗。理想情况下，检测策略应该是非侵入性的、快速的，并且易于在临床环境中部署。 循环中的游离DNA(CfDNA)是由正常细胞释放的，然而，在癌症患者中，有一部分 CfDNA来自肿瘤细胞(循环肿瘤DNA或ctDNA)。因为肿瘤细胞分裂的速度比 正常细胞、癌症患者通常因为坏死或凋亡而在血清中有高水平的cfDNA， 更大比例的cfDNA可归因于肿瘤。CfDNA中ctDNA片段的检测 为非特异性的、基于血液或尿液的筛查提供了一个极好的机会，称为“液体” 活组织检查“。癌症患者DNA中最容易检测到的变化之一是模式的变化 基因组甲基化是基因表达的重要控制机制。然而，有有限的 来自检测cfDNA增加的诊断信息，以及后来针对ctDNA和 这种DNA的癌症特异性改变。 在这里，我们建议使用固态纳米孔来检测高甲基化的胞嘧啶 CfDNA中鸟嘌呤(CpG)二核苷酸簇状岛(CGI)作为一种敏感和非特异性的检测技术 从血液或尿液样本中检测癌症。与蛋白质纳米孔一样，固态纳米孔允许 单分子测量与DNA链通过孔的移位导致独特的 电信号。序列将在甲基结合结构域(MBD)蛋白存在的情况下进行评估 选择性结合甲基化的CpG结构域和进一步扩增衍生的纳米孔 量化信号。在我们提出的方法中，电子签名代表了整个 分子，而不是单个核苷酸，用于检测高甲基化CGI作为癌症生物标记物。 拟议的工作得到三个技术目标(ToS)的支持。设置为1是为了确定灵敏度 每个CGI片段的甲基化CpG数目的方法。TO2将决定我们的能力 检测部分甲基化背景以上CGI高甲基化的方法 对照样本中的cfDNA。TO3将在一系列测试中确定该系统的可行性和敏感性 临床癌症模拟样本。 与以前开发的传统方法相比，我们提出的方法将是快速、灵敏、 并且与潜在的DNA序列无关，因此它可以用于癌症检测 靶标序列中的突变。这种基于固态的方法最终可以商业化为 简单易用，临床设置一般癌症检测和治疗进展诊断。

METHYL-SENTRY: Proposed feasibility study of a nanopore diagnostic tool to detect hypermethylated biomarkers in cell free DNA indicative of cancer state Early detection of cancer is correlated with improved outcomes and reduced morbidity due to more timely treatment. Ideally, detection strategies should be non-invasive, rapid, and easy to deploy in a clinical setting. Circulating cell-free DNA (cfDNA) is released by normal cells, however, in cancer patients a portion of cfDNA comes from tumor cells (circulating-tumor DNA or ctDNA). As tumor cells divide faster than normal cells, cancer patients typically have a high level of cfDNA in serum because of necrosis or apoptosis, with a greater proportion of cfDNA attributable to tumors. Detection of the ctDNA fraction of cfDNA presents an excellent opportunity for a non-specific, blood- or urine-based screening, referred to as a “liquid biopsy”. One of the most readily detected changes in the DNA of cancer patients is alterations in the pattern of genome methylation, a significant control mechanism of gene expression. However, there is limited diagnostic information from detecting an increase in cfDNA, and later approaches targeted ctDNA and cancer-specific alterations to this DNA. Here we propose the use of solid-state nanopores for detection of hypermethylated cytosine adjacent guanine (CpG) dinucleotide clustered islands (CGI) in cfDNA as a sensitive and non-specific technique for detection of cancer from blood or urine samples. As with protein nanopores, solid-state nanopores allow single molecule measurements with translocation of the DNA strand through the pore resulting in a unique electrical signal. Sequences will be evaluated in the presence of methyl binding domain (MBD) protein for selectively binding to methylated CpG domains and further amplification of nanopore derived quantification signal. In our proposed method, the electrical signature is representative of the entire molecule, instead of a single nucleotide, for detection of hypermethylated CGIs as a cancer biomarker. The proposed work is supported by three Technical Objectives (TOs). TO1 is to determine the sensitivity of the method for the number of methylated CpG per CGI fragment. TO2 will determine the ability of our method to detect an increase in hypermethylation of CGIs above the background of partially methylated cfDNA in a control sample. TO3 will determine the feasibility and sensitivity of this system in a series of clinical cancer mimic samples. In contrast to previously developed conventional methods, our proposed method would be fast, sensitive, and independent of the underlying DNA sequence so that it could be used for cancer detection regardless of mutations in the target sequence. This solid-state based method can ultimately be commercialized into a simple to use clinical setting general cancer detection and treatment progression diagnostic.