Switchable lanthanide luminescence is a novel proximity-based binary probe technology wherein two oligonucleotide probes are labelled either with lanthanide ion carrier chelate or light-absorbing antenna ligand. A highly luminescent complex is formed when the two label moieties are brought in close contact by adjacent hybridization of the labelled probes to a target sequence. Herein we report how hybridization distance between the labelled probes and single nucleotide mismatches in the target sequence affect to the luminescence intensity of the complex formed from the label moieties. The highest luminescence intensity was observed when the probes were hybridized to full matched target at a distance of 4-10 nucleotides, a fact that indicates the lanthanide complex requires a certain space to be formed. At optimal hybridization distance between the labelled probes the maximal discrimination efficiency between a full matched and a single mismatched target was 1400-fold. Furthermore, computer-based modelling of the luminescent complex formation was in good agreement with the experimental results. (C) 2015 Elsevier B.V. All rights reserved.