miRNA-Responsive 3D DNA walker nanoprobe for intracellular imaging, cell typing, and clinical diagnostics.

• Innovates miRNA detection with cholesterol-enhanced 3D DNA Walker, enhancing precision. • Uses micelle structure for nuclease protection, cutting false positives in samples. • Allows cancer cell autonomous miRNA imaging, no need for external cofactors. • Enables precise cancer subtype identification via multiple miRNAs, boosting accuracy. • Advances molecular diagnostics, key for early detection and personalized medicine. Achieving reliable miRNA detection and imaging remains a critical and formidable challenge. To address this challenge, we developed a three-dimensional (3D) DNA walker nanoprobe (DWN), derived from a multifunctional molecular beacon with a lipidic moiety. This innovative structure employs a cholesterol-conjugated multifunctional molecular beacon (Chol-MMB) assembled through hydrophobic interactions, thereby bypassing the cumbersome and inefficient process of immobilizing DNA strands onto rigid surfaces. Once the walker nanoprobe enters cells, the binding between the target miRNA and the nanoprobe triggers an intermolecular polymerization process, leading to a conformational change in DWN. This change liberates a walking strand, initiating the walking reaction. As a result, the 3D DNA walker nanoprobe illuminates the fluorophore, enabling precise quantitative analysis. The efficacy of the DWN was demonstrated by the limit of detection of 0.326 fmol for miR-21 with a reaction duration of 1 h. In addition, this strategy effectively differentiates various cells based on the specific miRNA. Further refinement of this method will lead to an efficacious miRNA-based platform for cell typing and liquid biopsy. [ABSTRACT FROM AUTHOR]