An innovative signal amplification strategy assisted by RNase H is described here for the detection of DNA targets in a universal fashion. A tailor-made RNA molecular beacon (RMB) conjugated with a fluorescence resonance energy transfer (FRET) pair (fluorophore and quencher) was designed, characterized, and combined with the employment of RNase H. Its performance is compared to that of other nucleases including Exonuclease III and T7 exonuclease. Fluorometry, performed best at excitation/emission wavelengths of 490/520 nm, gives an amazingly low detection limit of 23 fM for target DNA. The method was verified by the determination of human hemochromatosis (HFE) gene. It is perceived that the method represents a versatile tool for the detection of a wide range of targets. Graphical Abstract An RNase H-assisted signal amplification (RASA) method for the fluorometric assay of nucleic acids has been developed by using a unique RNA molecular beacon (RNA MB) conjugated with a fluorophore (F) and quencher (Q) pair for signal generation.
Keywords: DNA; Detection limit; Exonuclease III; Selectivity; Sensitivity; Signal amplification; T7 exonuclease.