Enhanced recognition of single-base mismatch using locked nucleic acid-integrated hairpin DNA probes revealed by...

Probe design is a critical parameter in successful DNA and RNA target detection. In this proof-of-concept study, we evaluated the single-base mismatch recognition power of surface immobilized and self-assembled stem-loop hairpin DNA oligonucleotide probes modified to contain locked nucleic acid residues (LNA-HP). The stiffness change in conjunction with the stem opening of the interfacial molecules before and after hybridization led to clear variations of the overall film thickness or miniaturized nanospot height, which could be directly measured using an atomic force microscopy (AFM) nanolithography technique. Particularly, LNA-HP achieved highly differentiable readouts between perfectly complementary and singly mismatched targets (discrimination ratio as high as 2 to 3), outperforming the selectivity of its linear and hairpin counterparts with no LNA modification.