A label-free and sensitive fluorescence strategy for screening ligands binding to poly(dA) based on exonuclease I-assisted background noise reduction

Abstract

A simple, rapid, label-free, and sensitive fluorescence strategy has been developed for screening the ligands binding to poly(dA) based on exonuclease I-assisted background noise reduction. In this strategy, we have designed 16-repeat deoxyribonucleic acids (A16) as a DNA probe and a double-stranded-chelating dye SYBR Green I (SG I) as a fluorescence dye. Exonuclease I (Exo I), a sequence-independent nuclease, was selected to digest the single-stranded DNA probe to minimize the background fluorescence signal. As a result, in the absence of the target molecular coralyne, A16 is digested by Exo I from its 3′ end. This leads to low background fluorescence due to the weak electrostatic interaction between SG I and mononucleotides (dA). On the other hand, the presence of coralyne can induce the single-stranded A16 to form the homo-adenine DNA duplex, and Exo I is inactive to this duplex structure, resulting in a remarkable fluorescence response. Upon background noise reduction, the sensitivity is improved significantly, with a detection limit of 3.5 nM, which is much lower than almost all the previously reported methods. Moreover, this method could extend the application to recognition interaction between ligands and functional nucleic acids and it could find wide applications in the screening of potential therapeutic molecules.