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.
Keywords: cholesterol-conjugated palindromic molecular beacon; circulating tumor DNAs (ctDNAs); nick-hidden hairpin-shaped DNA assembly; nuclease-resistant DNA nano-structure; single DNA probe-based strand displacement amplification in serum.