Peptide–DNA conjugate-assisted DNA cyclization for highly efficient and sensitive detection of furin†

Abstract

Furin, a critical regulatory protease, plays a crucial role in significant physiological processes such as oncogenic signaling pathways. Accurate detection of furin is essential for disease diagnosis and precise medical interventions. Herein, we leverage the proximity effect of the engineered peptide–DNA conjugate (PDC) to induce DNA cyclization, enabling efficient and sensitive detection of furin. The PDC is synthesized through chemical reactions that link peptides and DNA molecules, strategically structured with a peptide moiety containing the specific recognition and cleavage site for furin, sandwiched between two DNA sequences. Meanwhile, the proximity effect-mediated DNA hybridization of the PDC achieves the complementation of Padlock DNA, thereby ensuring circular DNA formation. Subsequently, the cyclized DNA serves as a template to initiate the rolling circle amplification reaction, generating numerous G-quadruplex structures that yield a fluorescence signal upon thioflavin T intercalation. While furin-catalyzed peptide cleavage disrupts the proximity effect, DNA cyclization and subsequent reactions are inhibited. The multifunctional PDC probe which can assist DNA cyclization facilitates furin recognition through peptide embedding, thus providing a new way for sensitive detection of furin, with a detection limit of 1.5 pM. Additionally, our method demonstrates efficacy in inhibitor determination, showing great potential across biomedical research, clinical diagnostics, and therapeutic development.