Enhanced Sensitivity in Oligonucleotide Probes via Terminal Excimer Formation

Fluorescence-based techniques are widely used to detect DNA mutations by monitoring changes in fluorescence signals that arise when DNA probes or dyes interact differently with mutated and normal DNA sequences. Such variations typically result from mismatched base pairing, altered melting temperatures, or changes in molecular interactions. This study aimed to investigate excimer fluorescence as a sensitive approach for distinguishing single-stranded and double-stranded DNA and for potential application in mutation detection. Excimer emission signals were analyzed in solution using fluorescence spectroscopy. Complementary oligonucleotides were labeled with pyrene fluorophores—one at the 5′ end of the probe strand and the other at the 3′ end of its complement. The proximity of the two pyrene groups upon hybridization was monitored to assess excimer formation. Hybridization of the complementary strands brought the two pyrene moieties into proximity, producing a strong excimer emission band at approximately 480 nm. This signal clearly differentiated double-stranded DNA from single-stranded forms. The system demonstrated high sensitivity to hybridization events and was suitable for monitoring DNA structural transitions such as denaturation and reassociation. Excimer fluorescence provides a reliable and sensitive method for detecting DNA hybridization and distinguishing between matched and mismatched sequences. The approach enables continuous monitoring of DNA interactions at low concentrations and offers promising potential for mutation and single-nucleotide polymorphism (SNP) detection in molecular diagnostics.