Self-assembled Four-color Fluorescent Probe For Ratiometric And Multiplexed Detection Of Biomolecules

Nucleases hydrolyze the phosphodiester bonds in nucleic acid backbone through an endonucleolytic mechanism,and they play important roles in several cellular processes including DNA replication,recombination,repair,genotyping,gene mapping,and molecular cloning.A number of methods have been developed for the detection of nuclease activity,including gel electrophoresis,high performance liquid chromatography,enzyme-linked immunosorbent assay,molecular beacon based fluorescent assay,surface plasmon resonance and colorimetric methods.Nevertheless,these methods are usually time-consuming and laborious with the involvement of radioactive substances and the complicated synthesis of conjugated nanoparticles,and some of them are unable to distinguish multiple targets.Therefore,the development of new methods for multiplexed detection of nucleases is highly desired.Nanostructures formed by DNA self-assembly are either static with wellcontrolled physicochemical properties or dynamic with the capability to reconfigure upon external stimuli.So far,a variety of multi-dimensional shapes have been constructed for biocomputing,sensing,biocatalysis,nanorobotics,drug delivery,and plasmonic devices.On the basis of specific complementary Watson-Crick base pair hybridization,dyes and nanoparticles can be anchored at the predefined positions of DNA nanostructures,allowing for fluorescence resonance energy transfer(FRET)-based optical detection.The DNA-based FRET has found wide applications in nanoscale structural analysis,biosensing,theranostics.The DNA tetrahedral nanostructure is considered to be an excellent DNA framework candidate for the assembly of functional nanodevices because of its well-defined three-dimensional structure and convenient nanoassembly.In this research,we develop a new four-color fluorescent probe for ratiometric detection of multiple nucleases based on the integration of FRET with DNA tetrahedral nanostructure.The core DNA tetrahedral nanostructure can be readily assembled with four designed 53-mer strands with a simple annealing process.The probe is constructed by the dye-labeled DNAs bearing four fluorophores: diethylaminocoumarin(DEA),carboxyfluorescein(FAM),Texas Red,and Cy5.The intact four-color fluorescent probe is designed as a multistep FRET process with DEA as a donor,FAM and Texas Red as an acceptor/donor,and Cy5 as an acceptor.When DEA is excited,the energy can be transferred to Cy5 through FAM and Texas Red,leading to fluorescence emission of DEA,FAM,Texas Red and Cy5.This four-color fluorescent probe contains three cleavage sites of XhoI,HindIII and KpnI,In the presence of specific enzyme,it can cleave the appropriate site,inducing the fluorescence changes of different dyes and consequently allowing for ratiometric detection of multiple nucleases.This probe consists of four fluorescent dyes connected by DNA tetrahedral nanostructure with the involvement of multistep FRET processes.This four-color fluorescent probe can be excited by a single wavelength and generates four emission signals simultaneously,and it can unambiguously distinguish multiple nucleases including KpnI,HindIII and XhoI,with distinct advantage of simplicity over the conventional FRET probes(each comprises a single donor/acceptor pair)for multiplexed assay.In addition,this four-color fluorescent probe exhibits good selectivity against other nucleases,and it can be applied to screen the inhibitors of nucleases.Importantly,this four-color fluorescent probe can be structurally modified with other cleavable sites for various nucleases and ribonucleases,with wide applications in chemical biology,biomedical research,and clinical diagnosis.