Nucleic Acid FRET Sensing of Hydrogen Peroxide in Live Cells Using a Boronic Acid Nucleobase Surrogate

For activity-based sensing, where probes detect biological analytes through chemical reactivity, nucleic acid (NA) scaffolds are seldom employed. Herein, we present the first all-in-one NA sensor (KR28) that is activated by H2O2 as a molecular trigger to foster a turn-on FRET response in human serum and in the nucleus of live cells. The 28-mer 2ʹ-OMe-XNA hairpin (HP) KR28 features a boronic acid nucleobase surrogate (BA6HI) that serves as the H2O2 sensing element and is embedded into KR28 using an on-strand Aldol condensation approach. Ipso-hydroxylation of the BA6HI surrogate mediated by H2O2 furnishes an emissive phenolic product (PhOH6HI, λex/λem = 390/490 nm) that serves as the FRET donor to a thiophene surrogate acceptor (Th6HI, λex/λem = 530/580 nm) for a FRET efficiency of 95%. The BA6HI-modified NA scaffolds match the sensitivity of free boronic acid dyes for H2O2 (~ 150 nM) and localize in the nucleus of live cells for nuclear H2O2 FRET detection. Our work expands the function of NA scaffolds beyond their use as molecular recognition elements and demonstrates their potential to serve as activity-based sensors of ROS to probe the relationships between nuclear oxidative stress and disease states.