DNA Nanoruler‐Assisted Linearization for Long‐Chain Nucleic Acids Analysis

ABSTRACT Long‐chain nucleic acids (LCNAs) are increasingly important as diagnostic biomarkers, yet their quantitative detection is often hindered by compact conformations that limit probe accessibility. Here, we report a DNA nanoruler‐assisted linearization strategy that mechanically regulates LCNA conformation by employing a rigid DNA tetrahedral framework (DTF) dimer as a nanoruler. This approach anchors the target termini at a predefined nanoscale spacing (∼10–15 nm), imposing defined spatial constraints that stretch and pre‐open secondary structures, leading to linearization and enhanced site accessibility. In homogeneous assays, this strategy delivers robust signal enhancement across LCNAs with diverse structures. Integrated into an electrochemical platform, it achieves LODs of 1 copy/µL for the E and N genes and 10 copies/µL for ORF1ab, with linear ranges of 10–10 5 copies/µL for E gene and 10 2 –10 5 copies/µL for both N gene and ORF1ab from SARS‐CoV‐2. Furthermore, when incorporated into a fluorescence chip, this method enables high‐throughput analysis of circulating cfDNA (ALU115) for prostate cancer discrimination with an AUC of 90.35%. Collectively, we establish a DNA nanoruler‐based conformational regulation strategy as a structure‐tolerant approach for high‐performance LCNA detection in both electrochemical and fluorescent modes.