DNA nanomachines based on gold nanoparticles (AuNPs) are promising platforms for intracellular sensing of microRNAs (miRNAs), which are biomarkers involved in a wide range of diseases. However, achieving robust performance under intracellular conditions remains challenging. In this study, we investigated the effect of locked nucleic acid (LNA) modification on an entropy-driven DNA-AuNP nanomachine designed for miRNA detection. By incorporating LNA into probe sequences, we evaluated signal generation, sensitivity, and selectivity under cytosol-mimicking conditions. Consequently, appropriately designed LNA modifications substantially restored nanomachine functionality under intracellularly relevant conditions. In contrast, excessive or unfavorably positioned LNA incorporation led to increased nonspecific background signals. Under optimized conditions, the LNA-modified nanomachine achieved a detection limit of approximately 10 pM for miRNA-21 with high sequence specificity. Furthermore, when delivered into MCF-7 cancer cells using a transfection reagent, the LNA-modified DNA-AuNP nanomachine showed a higher fraction of fluorescence-positive cells compared with the unmodified system. This study clarifies the critical role of probe modification in the design of DNA-AuNP nanomachines operating within living cells and presents an effective strategy for realizing live-cell miRNA imaging based on molecular robotics concepts.

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