RESUMO
DNA nanoswitches can be designed to detect unlabelled nucleic acid targets and have been shown to discriminate between targets which differ in the identity of only one base. This paper demonstrates that the fluorescent base analogue 2-aminopurine (AP) can be used to discriminate between nanoswitches with and without targets and to discriminate between matched and mismatched targets. In particular, we have used both steady-state and time-resolved fluorescence spectroscopy to determine differences in AP environment at the branchpoint of nanoswitches assembled using complementary targets and targets which incorporate single base mismatches.
Assuntos
2-Aminopurina/química , Pareamento Incorreto de Bases , DNA/química , Sondas de DNA/química , Transferência Ressonante de Energia de Fluorescência , Nucleotídeos/genética , Espectrometria de Fluorescência/métodosRESUMO
We present a new type of DNA switch, based on the Holliday junction, that uses a combination of binding and conformational switching to enable specific label-free detection of DNA and RNA. We show that a single RNA oligonucleotide species can be detected in a complex mixture of extracted cellular RNA and demonstrate that by exploiting different aspects of the switch characteristics we can achieve 30-fold discrimination between single-nucleotide mismatches in a DNA oligonucleotide.