Construction Of Functional Framework Nucleic Acid And Its Application In Cell Imaging

Cancer biomarkers are inextricably linked to the stage of cancer and can therefore be used for cancer screening and clinical diagnosis.At present,most of the detection methods of cancer markers have problems such as complicated operations,high cost,or low sensitivity.Therefore,the construction of effective cancer marker detection methods has become the focus of cancer risk assessment,early diagnosis,intra-disease monitoring and postoperative monitoring.Emerging structural DNA nanotechnology provides new avenues for the design and construction of self-assembled nanostructures,with framework nucleic acids（FNAs）such as DNA tetrahedral（TDN）and DNA cube attracting attention.First,through the precise programmability of its size and shape,reconfigurable FNAs can be built.Second,its precise addressability enables the delicate loading of guest objects to construct biological vehicles.In addition,the FNAs have a precisely measurable and adjustable internal space,and are an excellent reactor loading bracket,providing reliable protection for the reaction with an excellent domain limiting effect.Based on the powerful and structurally controllable FNAs,this manuscript synthesizes nanodevices with different application functions（carrier function,signal amplification function,motor function）,and carries out high-sensitivity and high-specificity analytical detection and cell imaging of a variety of cancer markers.The specific research content is as follows:（1）A series of high-order DNA tetrahedral nanocarrier structures（mono/di/tri-TNC）with high purity and good yield were prepared by one-pot boiling and size exclusion chromatography purification method.The individual DNA tetrahedral unit structures are connected by long chains,and the nanostructures of mono/di/tri-TNC are observed by atomic force microscopy.Precise spatial modification is performed on one edge of the tri-TNC unit structure to achieve precise and controllable pricing state assembly of molecular beacons,antisense nucleic acid chains,and Au NPs.（2）Investigate the biological application of tri-TNC for simultaneous detection of multiple miRNAs in multiple systems.In homogeneous media,tri-TNC can detect three identical miRNAs and three different kinds of biomarkers without interfering with each other.In serum samples,by analyzing the differences between the three miRNAs in the serum,healthy people,patients with prostate enlargement,and patients with prostate cancer were successfully distinguished.In cell experiments,molecular beacons are transported intracellularly by tri-TNC to achieve in situ imaging of cells and successfully distinguish between many different cell lines.（3）Two finely catalyzed hairpin components are encapsulated in the internal cavity of the DNA cube to construct an enzymatic limit-free amplifier（L-DNC）for detecting intracellular miRNA-21.L-DNC can be smoothly uptaken by cells,and the distinction between A549 and QGS-7701 cells is achieved by miRNA-21.In in vitro experiments,the maximum reaction rate of L-DNC was increased by a factor of 14.34compared to free-CHA,with a detection limit of 40 p M.The local concentration of the L-DNC probe is theoretically calculated to be 5000times higher than that of free-CHA,which is mutually corroborated with the actual test results.（4）Two mutually coordinated displacement assemblies are precisely assembled inside a DNA cube,with endogenous m RNA as fuel,to build a self-powered captive domain signal amplifier（RL-cube）for detecting Let-7a.The RL-cube has a reaction rate of approximately 4 times higher than conventional chain-displacement probes,with a reaction saturation time of only 7 min and a detection limit of 7.57 p M.In the cell experiment,the accurate differentiation of different cell lines was achieved,and the content of Let-7a in MCF-7 was calculated to be 9.64 n M,providing a data reference for the quantification of intracellular miRNA.（5）The surface of UiO-66-N₃ is half covered with a 5 nm-thick gold shell,and half of the modification can specifically identify the TDN of three membrane proteins.Thus,a new nanomotor material（JNM@TDN）with both photothermal and specific recognition properties is constructed.JNM@TDN has good morphology,high yield,good dispersibility,low cytotoxicity,and can be effectively uptaken by cells.Powered by an 808nm laser and TDN as the"navigation",it realizes the accurate identification of multiple membrane proteins on different cell lines and images in situ on the cell membrane.