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 represent an attractive platform to enhance biocompatibility and permit ratiometric analyte detection through the precise control of distance and orientation of donor/acceptor probes within the duplex framework for a turn-on FRET response. Herein, we present the first all-in-one NA FRET sensor (KR28) that is activated by H₂O₂ as a molecular trigger in human serum and in the nucleus of live cells. The 28-mer 2'-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. <i>ipso</i>-Hydroxylation of the BA6HI surrogate mediated by H₂O₂ furnishes an emissive phenolic product (PhOH6HI, λ_ex/λ_em = 390/490 nm) that serves as the FRET donor to a thiophene surrogate acceptor (Th6HI, λ_ex/λ_em = 530/580 nm) for a FRET efficiency ∼ 95%. The BA6HI-modified NA scaffolds can detect H₂O₂ in the low nM regime 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 through noncovalent affinity interactions and demonstrates their potential to serve as activity-based sensors of ROS to probe the relationships between nuclear oxidative stress and disease states.