The Study Of Biosensing And Molecular Logic Computing Based On DNA Triangular Prism Nanoprobe

Owing to nanostructures have the capacity of precisely controllable and highly addressable,excellent biocompatibilty and biostability,high cell membrane permeablity and controlled drug release property.DNA nanostructures have been widely applied in biosensing,cellular imaging,drug delivery and assembling new nanomaterials,such as liposomes,protein synthesis.In this thesis,two functional DNA nanostructures were constructed based on the DNA triprism and used for further biological research.The main research contents are summarized as follows:(1)Based on the self-assembly strategy of DNA single strand tiles,we have assembled a three-dimensional(3D)minimal-DNA triangular nanoprism with high symmetry and multiple binding sites.Native polyacrylamide gel electrophoresis(PAGE)were carried out to verify the self-assembly of the DNA triangular nanoprism.Dynamic light scattering(DLS)charactered the size of the DNA triangular prism to be(16.0±2.0)nm,and the atomic force microscope(AFM)scan analyzed its height at(1.3±0.3)um.Also,the DNA triangular nanoprism could keep its structure intact for more than 6 h in 10%FBS,which indicated that the DNA triangular nanoprism have excellent biostability and could be applied in complex biological microenvironment.(2)Based on the split aptamer and the DNA triangular prism(TP),we have developed a DNA TP nanoprobe for adenosine triphosphate(ATP)sensing in living cells.In the in vitro assay,the fluorescence emission ratio(FA/FD)was found to be linear with the concentration of ATP in the range of 0.03-2 mM,and the nanoprobe was highly selective toward ATP.For the strong protecting capability to nucleic acids from enzymatic cleavage and the excellent biocompatibility of the TP,the DNA TP nanoprobe exhibited high cellular permeability.Meanwhile,the intracellular imaging experiments indicated that the DNA TP nanoprobe could effectively detect ATP and distinguish among changes of ATP levels in living cells.More importantly,using of the split aptamer and the FRET-off to FRET-on sensing mechanism could efficiently avoid false-positive signals.(3)We developed a three-dimensional(3D)DNA-based logic gate nanomachine.It can not only target overexpressed cancer cell biomarkers,but also carry out "AND" Boolean operation on the surface of the target cell membrane and output "ON" signal.Compared to double-stranded DNA(dsDNA)-based molecular circuits,this 3D DNA-logic gate triangular prism(TP)nanomachine showed better computation performance,enabling better recognition and molecular targeting.