Advances in Double‐Stranded DNA Targeting Technologies

Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.