Design And Application Of Framework Nucleic Acid Fluorescent Nanoprobes

The study of complex events in living systems requires the simultaneous detection and analysis of multiple targets in complex samples.However,the limited number of recognizable fluorophores in the visible range leads to limitations in the ability of multiplexing.DNA,as a molecular carrier of hereditary information in nature,can encode important biological macromolecules in living systems through the genetic code.In recent years,the development of DNA nanotechnology has enabled us to construct a variety of different functional nanostructures with the help of DNA,and these functional nanostructures can be widely used in life sciences,biocomputing and bioimaging.In this work,the construction and application of framework nucleic acid fluorescent nanoprobes were realized by combining DNA nanotechnology and fluorescence technology to investigate the luminescent behavior and fluorescence anisotropy of fluorescent molecules.Details are as follows:First,fluorescent excimer detection of transient conformational changes in DNA with intercalative dye,K21.DNA conformational changes are important in regulating gene expression and mediating drug-DNA interactions.However,direct detection of transient DNA conformational changes is challenging due to the dynamic nature of this process.Here,we propose a label-free real-time fluorescence method for the detection of transient conformational changes in DNA of different lengths and structures.By investigating the luminescence behavior of K21 after interaction with DNA,we found that K21 can aggregate on the DNA surface and form transient excimer aggregates,and the fluorescence emission ratios of K21 monomer and excimer aggregates correlate with the stability of transient conformation of DNA structures.This method can effectively identify and distinguish multiple DNA nanostructures with highly similar but structurally different(such as i-motif,G-quadruplex,double-stranded DNA with different number and position of base mismatches).Also,we use this method to identify different topologies of the same plasmid,and the results show that we can effectively partition DNA nanostructures of the same sequence and different topologies.This method provides a label-free fluorescence strategy to detect transient conformational changes in DNA structures and has the potential to reveal transient genomic processes in living cells.Second,effect of DNA tetrahedra(TDN)on the fluorescence anisotropy of intercalators.The fluorescence anisotropy of intercalator has important applications in the fields of nucleic acid analysis,fluorescent probe construction and biosensing,and there are few studies on the fluorescence anisotropy of intercalators based on framework nucleic acids.Here,we constructed a series of TDN fluorescence anisotropic probes by precisely regulating the size of TDN through DNA sequence design,combined with SYBR Green I,DAPI and Di YO-1,and investigated the effect of TDN size on the fluorescence anisotropy of the probes.The results showed that for SYBR Green I and DAPI,the fluorescence anisotropy of the probes increased with the increase of TDN size.For Di YO-1,the fluorescence anisotropy of the probe decreased and then increased with the increase of edge length of TDN.This study shows that the regulation of fluorescence anisotropy of fluorophore can be achieved by precisely regulating the TDN size,and the fluorescence anisotropy probe with framework nucleic acid as a carrier is expected to be a new and excellent probe.Third,encoding fluorescence anisotropy barcodes using framework nucleic acids.Fluorescence anisotropy(FA)has great potential for multiplexed analysis and imaging of biomolecules because it can effectively distinguish fluorophores with overlapping emission spectral features.However,its sensitivity to environmental changes hinders its widespread applications in multicolor imaging.Here,inspired by fluorescent proteins,we designed a fluorescence anisotropy DNA framework(FAF)by scaffolding fluorophores in a fluorescent protein-like microenvironment.We found that the stability of FA of fluorophore was significantly improved due to the sequestration effects of FAF.When fluorophores are placed at different positions on the FAF,their FA can be finely tuned,which is similar to the spectral shift of a protein-bound fluorophores.The high programmability of the FAF allows us to design a series of spectrally encoded FA barcodes and use them for multiplexed detection of nucleic acids and multiplexed labeling of living cells.The FAF system provides a new approach for multiplexed FA probe design.