Stimuli-Induced Upgrade of Nuclease-Resistant DNA Nanostructure Composed of a Single Molecular Beacon for Detecting Mutant Genes

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.