DNA apurinic/apyrimidinic site (AP site) has received much attention due to its importance in disease evolution and development of selective genetic drugs. Fluorescence technique has been widely employed in identifying the AP sequence environment using fluorophores as the AP site-targeting probes. However, the sequence effect on the AP site binding of a ligand should be fully understood for illustrating the binding behavior. Triplex-forming oligonucleotide (TFO) can bind with its partner ds-DNA via specific hydrogen bonding interactions different from the canonical Watson-Crick pattern to form a triplex DNA structure . Due to its wide bioactivity, triplex has been recently investigated using small molecules as the structure identifiers and tuners. In this work, we spectroscopically characterized a novel fluorescent benz[ e ]indolium probe of BIFS and found that BIFS can selectively target the AP site and its fluorescence is significantly enhanced only when the TFO strand binds to the AP site-containing duplex DNA. The TFO binding restricts the non-radiative twist relaxation process of the excited state BIFS and results in the emissive BIFS. Because BIFS alone in aqueous solution is non-fluorescent, this selective lighting-up emission is advantageous for developing a practical sensor to target the triplex AP site with a weak fluorescence background. This selective recognition of the AP site by the lighting-up fluorescent probe would find wide applications including fluorescently evaluating the triplex-based small molecular binders.
Apurinic/apyrimidinic endonuclease 1 (APE1) selectively cleaves the apurinic/apyrimidinic site (AP site) in DNA, playing a critical role in base excision repair and genomic stability maintenance. Aberrant APE1 expression has been linked to numerous diseases, including cardiovascular disorders, neurological conditions, and various cancers. However, existing methods for detecting trace levels of APE1 remain suboptimal for certain applications. To address this limitation, we developed an innovative biosensing platform for ultrasensitive APE1 detection by integrating APE1-specific site recognition with hybridization chain reaction (HCR)-based signal amplification, enabling enzyme- and label-free bioassays. In this system, APE1 recognizes and cleaves the AP site-containing hairpin (HP) probe, releasing a single-stranded HCR initiator that triggers cascaded HCR amplification. Owing to the high efficiency of HCR, this method achieves ultrahigh sensitivity, with a calculated detection limit of 1.0 × 10−8U/mL. Furthermore, the biosensor demonstrates robust performance in cell lysates and is applicable for screening and evaluating APE1 inhibitors.
Constructing precise delivery systems to enhance the antitumor efficacy of drugs represents a pressing clinical practice need. The characteristics of suitable physiological stability, flexible tunable size, and highly programmable architecture make functional DNA scaffolds an ideal solution to this requirement. Here, we designed DNA oligonucleotide probes Y 1 , Y 2 and Y 3 to construct a smart-responsive Y-shaped DNA scaffold drug conjugate (sYDD). This sYDD incorporates two AS1411 aptamers, an apurinic/apyrimidinic (AP) site, a B-cell lymphoma-2 (Bcl-2) antisense oligonucleotide (ASO) and a doxorubicin (DOX)-carrying sequence. The Bcl-2 ASO and DOX within sYDD could be responsively released in tumor cells that exhibited high expression of apurinic/apyrimidinic endonuclease 1 (APE1) and microRNA-21 (miR-21). And the in vitro and in vivo experiments demonstrated that sYDD exhibited substantial antitumor efficiency. Moreover, the flow cytometry and RNA sequencing results proved that the proposed sYDD effectively activated immune responses. This strategy provides an alternative approach for designing synergistic chemo-gene drug conjugates, which facilitates the development of precision tumor treatment modalities.
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