Construction of Tetrahedral DNA-Quantum Dot Nanostructure with the Integration of Multistep Forster Resonance Energy Transfer for Multiplex Enzymes Assay

Three-dimensional (3D) DNA scaffolds with well-defined structure and high controllability hold promising potentials for biosensing and drug delivery. However, most of 3D DNA scaffolds can detect only a single type of molecule with the involvement of complex logic operations. Herein, we develop a 3D DNA nanostructure with the capability of multiplexed detection by exploiting a multistep Forster resonance energy transfer (FRET). The tetrahedron-structured DNA is constructed by four oligonucleotide strands and is subsequently conjugated to a streptavidin-coated quantum dot (QD) to obtain a QD-Cy3-Texas Red-Cy5 tetrahedron DNA. This QD-Cy3-Texas Red-Cy5 tetrahedral DNA nanostructure has well-defined dye-to-dye spacing and high controllability for energy transfer between intermediary acceptors and terminal acceptors, enabling the generation of multistep FRET between the QD and three dyes (i.e., Cy3, Texas Red, and Cy5) for simultaneous detection of multiple endonucleases and methyltransferases even in complex biological samples as well as the screening of multiple enzyme inhibitors.