Modular DNA Tetrahedron Nanomachine-Guided Dual-Responsive Hybridization Chain Reactions for Discernible Bivariate Assay and Cell Imaging

Engineering of multivariate biosensingand imaging platforms involvedin disease plays a vital role in effectively discerning cancer cellsfrom normal cells and facilitating reliable targeted therapy. Multiplebiomarkers such as mucin 1 (MUC1) and nucleolin are typically overexpressedin breast cancer cells compared to normal human breast epitheliumcells. Motivated by this knowledge, a dual-responsive DNA tetrahedronnanomachine (drDT-NM) is constructed through immobilizingtwo recognition modules, MUC1 aptamer (MA) and a hairpin H1* encodingnucleolin-specific G-rich AS1411 aptamer, in two separate vertexesof a functional DT architecture tethering two localized pendants (P-M and P-N). When drDT-NM identifiablybinds bivariate MUC1 and nucleolin, two independent hybridizationchain reactions (HCRM and HCRN) as amplificationmodules are initiated with two sets of four functional hairpin reactants.Among them, one hairpin for HCRM is dually ended by fluoresceinand quencher BHQ1 to sense MUC1. The responsiveness of nucleolin isexecuted by operating HCRN utilizing another two hairpinsprogrammed with two pairs of AS1411 splits. In the shared HCRN duplex products, the parent AS1411 aptamers are cooperativelymerged and folded into G-quadruplex concatemers to embed Zn-protoporphyrinIX (ZnPPIX/G4) for fluorescence signaling readout, thereby achievinga highly sensitive intracellular assay and discernible cell imaging.The tandem ZnPPIX/G4 unities also act as imaging agents and therapeuticcargos for efficient photodynamic therapy of cancer cells. Based on drDT-NM to guide bispecific HCR amplifiers for adaptivebivariate detection, we present a paradigm of exquisitely integratingmodular DNA nanostructures with nonenzymatic nucleic acid amplification,thus creating a versatile biosensing platform as a promising candidatefor accurate assay, discernible cell imaging, and targeted therapy.

Automated summary

Engineering multivariate biosensing and imaging platforms is crucial for distinguishing cancer cells from normal cells and facilitating targeted therapy. In this study, a dual-responsive DNA tetrahedron nanomachine was constructed, incorporating recognition modules for MUC1 and nucleolin. This nanomachine effectively detected bi-variate MUC1 and nucleolin, triggering amplification modules for sensitive intracellular assay and cell imaging. Additionally, the nanomachine served as an imaging agent and therapeutic cargo for photodynamic therapy of cancer cells, showing great potential for accurate assay, discernible cell imaging, and targeted therapy.