Multiplex Detection of Endonucleases by Using a Multicolor Gold Nanobeacon

A highly sensitive and selective assay based on a novel enzyme-responsive multicolor gold nanobeacon has been developed for the multiplex detection of endonucleases, a group of very important nucleases. The nanobeacon takes advantage of the high specificity of DNA cleavage reactions combined with the unique fluorescence-quenching property of gold nanoparticles (AuNPs). To prepare the nanobeacon, three hairpin DNA reporters, each labeled at the 5' terminus with a fluorescent dye (i.e., fluorescein amidite (FAM), carboxy-X-rhodamine (ROX), cyanine dye (Cy5)), that respond to one of three different endonucleases are co-assembled at the surface of AuNPs (15 nm). This assembly brings the dyes into very close proximity with the AuNP, which leads to significant quenching of the fluorescence due to the nanosurface energy-transfer (NSET) effect. When the nanobeacon is exposed to the targeted endonucleases, specific DNA cleavage occurs and pieces of DNA fragments are released from the AuNP surface along with the fluorescent dye, which results in the fluorescence recovery that provides the basis for a quantitative measurement of endonuclease activity. Three endonucleases, namely HaeIII, EcoRI, and EcoRV, were studied as the proof-of-concept analytes. These endonucleases in homogeneous mixture solutions were simultaneously quantified by the proposed assay with high sensitivity and specificity. The limits of detection obtained were in the range of 5.0 x 10(-4) U mL(-1) to 1.0 x 10(-3) U mL(-1) of endonuclease; these limits are at least 100 times more sensitive than the previously reported endonuclease assays. Endonuclease inhibitors impair the DNA cleavage, so it is anticipated that the present method has great potential for screening inhibitors of endonucleases. To demonstrate this application, the inhibitory effects of certain anticancer drugs on HaeIII, EcoRI, and EcoRV activities were studied. The present protocol proved to be sensitive, reliable, and easy to carry out.