College of Chemical Engineering, Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350108, China.
Hunan Provincial Key Laboratory of Phytohormones and Growth Development, College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China.
ACS Sens. 2021 Nov 26;6(11):4029-4037. doi: 10.1021/acssensors.1c01423. Epub 2021 Nov 3.
As a kind of cell-free DNA in the bloodstream liberated from tumor cells, circulating tumor DNAs (ctDNAs) have been recognized as promising biomarkers in the field of early cancer diagnosis. However, robust, sensitive, and accurate detection of ctDNA in serum remains extremely challenging, especially toward the mutant KRAS gene, one of the most frequently mutated genes. Although DNA oligonucleotides as emerging practical signaling materials have been developed as sensitive and accurate tools, some intrinsic defects need to be overcome, such as fragility in complex biological environments. In this work, on the basis of the hydrophilicity-promoted assembly, a core/shell DNA nanostructure (DNS-MB) probe is constructed from only one hairpin-shaped probe (cholesterol-modified palindromic molecular beacon, Chol-PMB) for the amplification detection of KRAS mutation in serum without the need for any auxiliary probe. Chol-PMB is designed to recognize target DNA and serve as a polymerization primer and template, and thus target species can initiate polymerization-based strand displacement amplification (SDA). Moreover, target DNA is able to induce further aggregation of DNS-MB particles due to the enzymatic cross-linking effect, leading to a structural upgrade. The DNS-MB probe exhibits a detection limit of 50 fM and a wide quantitative range (from 50 fM to 160 nM). In addition, single nucleotide polymorphisms can be discriminated, such as mutant KRAS G12D (KRAS-M), providing a desirable platform for screening ctDNAs. More excitingly, because the termini of DNA components are hidden inward from nuclease attack, DNS-MB circumvents a false-positive signal even in freshly sampled serum and is suitable for application in the complex biological milieu. As a proof of concept, the DNS-MB probe is expected to provide useful insight into the development of simple and degradation-resistant DNA probes for substantially amplified detection of ctDNAs in complex serum, showing potential applications in the field of early tumor diagnosis.
作为一种从肿瘤细胞中释放到血液中的无细胞 DNA，循环肿瘤 DNA（ctDNA）已被认为是早期癌症诊断领域有前途的生物标志物。然而，在血清中稳健、灵敏和准确地检测 ctDNA 仍然极具挑战性，特别是针对突变 KRAS 基因，这是最常突变的基因之一。尽管 DNA 寡核苷酸作为新兴的实用信号材料已经被开发出来作为灵敏和准确的工具，但仍需要克服一些内在缺陷，例如在复杂的生物环境中的脆弱性。在这项工作中，基于亲水性促进的组装，从一个发夹状探针（胆固醇修饰的回文分子信标，Chol-PMB）构建了一种核/壳 DNA 纳米结构（DNS-MB）探针，用于在无需任何辅助探针的情况下扩增检测血清中的 KRAS 突变。Chol-PMB 被设计用来识别靶 DNA 并作为聚合引物和模板，因此靶物质可以启动基于聚合的链置换扩增（SDA）。此外，由于酶交联效应，靶 DNA 能够诱导 DNS-MB 颗粒进一步聚集，导致结构升级。DNS-MB 探针的检测限为 50 fM，定量范围很宽（从 50 fM 到 160 nM）。此外，还可以区分单核苷酸多态性，例如突变 KRAS G12D（KRAS-M），为筛选 ctDNA 提供了一个理想的平台。更令人兴奋的是，由于 DNA 组件的末端从核酸酶攻击中向内隐藏，DNS-MB 即使在新采集的血清中也能避免假阳性信号，并且适用于复杂的生物环境。作为概念验证，DNS-MB 探针有望为简单且抗降解的 DNA 探针的开发提供有用的见解，用于在复杂的血清中进行实质性扩增检测 ctDNA，在早期肿瘤诊断领域具有潜在的应用前景。
