A novel single-molecule telomere characterization technology for analyzing cancer

描述（由申请人提供）：单端粒特异性（TTAGGG）n束长度和不稳定性不能使用当前的方法进行全球测量；这样做的能力将是一个重要的，变革性的补充，用于分析癌症中关键端粒丢失和端粒延伸事件的工具库。细胞中最短的端粒或最短端粒的一小部分将决定衰老、细胞凋亡或基因组不稳定的发生；单（TTAGGG）n束对端粒的功能以及端粒磨损和功能障碍的生物学效应至关重要。端粒丢失、断裂、融合和重新连接在癌症中高度增加，但目前在分子水平上检测和测量这些突变事件的方法有限且低通量。我们建议在此开发的技术将允许对端粒长度和不稳定性进行定量、单等位基因分辨率测量，从而前所未有地深入了解端粒丢失、端粒断裂/重新连接以及端粒酶或ALT依赖的端粒伸长在癌变中的作用。包括对介导这些过程的分子事件的机制见解以及对这些方法的潜在预后和肿瘤分层适用性的翻译见解。在这种方法中，

DESCRIPTION (provided by applicant): Single-telomere-specific (TTAGGG)n tract lengths and instabilities cannot be measured globally using current methods; the ability to do so would be a major, transformative addition to the arsenal of tools for analyzing critical telomere loss an telomere elongation events in cancer. The shortest telomere or a small subset of the shortest telomeres in a cell will determine the onset of senescence, apoptosis, or genome instability; single (TTAGGG)n tracts are crucial for the function of telomeres and the biological effects of telomere attrition and dysfunction. Telomere loss, breakage, fusion, and rejoining are highly elevated in cancer, but current methods for detecting and measuring these mutational events at the molecular level are limited and low-throughput. The technology we propose to develop here would permit quantitative, single- allele-resolution measurements of telomere length and instability, enabling unprecedented insights into the role(s) telomere loss, telomere breakage/re-joining, and telomerase or ALT dependent telomere elongation play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the methods. In this method, input genomic DNA is labeled with fluorescent dyes specific for (TTAGGG)n sequences and for linked subtelomeric DNA. The labeled individual DNA fragments are linearized (stretched) and imaged in the nano-channels of Bionano Genomics system at very high throughput. The lengths of the telomere are measured accurately and the distances between probes in the subtelomere region are determined accurately to infer the identity of the telomere. Our goals for this R21 study are to establish feasibility for (1) high-throughput single-molecule detection and quantitation of (TTAGGG)n tracts in genomic DNA samples and (2) subtelomere probe development and efficient co-labeling of telomeres and subtelomeres in the context of total genomic DNA, including conversion of the labeled DNA to double- stranded DNA suitable for nano-channel analysis; and (3) proof-of-principle results for the technology and its applicability for cancer research using data generated and analyzed for a normal and a cancer cell line. There are significant technical challenges inherent in this early-stage technology development project, but the extraordinary payoff for cancer research will be a high-throughput ability to probe mechanisms of telomere length regulation and telomere mutation at single-telomere resolution, in small (ultimately single-cell) samples of both dividing and non-dividing cells.