Designed Tetrahedral DNA Nanostructure And Its Applications In Cells Labelling

Tetrahedron DNA nanostructure is an excellent DNA nanostructures. By precisely designing the DNA sequences,single strand DNA hybridize with each other to form a three-dimensional DNA nanostructure. There are many advantages for TDNs, like structural controllability, biological compatibility and cell membrane permeability, which make it as an excellent nano-sensor platform in biological molecules detection and biomedical research. Now, nano-sensor has been a major way for biological molecules detection. For the importance of mRNA detection in biological research, diseases treatment and cells labelling, we design an mRNA detecting model for cell special labelling in tissue.In this paper, we firstly studied the cell uptake of TDNs in two cell lines and hippocampus neuron; Then we delivered TDNs to the brain tissue for studying cell uptake by mean of pressure microinjection and in vivo electroporation; In the end, combining TDNs with mRNA probes, we designed a mRNA detection model based on TDNs, which we called TDNs-flare, for cell special labelling in central nervous system.Firstly, we choose the TDNs with side length of 20 bp. After the assembly, characterization and purification of the TDNS, we studied the cell uptake of TDNs in Hela cell and PC 12 cell. It turn out that the two cell lines could efficiently absorb TDNs after we incubated cells with them for 12h; Then we cultivated the hippocampus neuron and confirmed them by immunofluorescence. Incubating hippocampus neuron with TDNs for 12 h, it came out that hippocampus neuron can also efficiently take in TDNs, which was help for research and application of TDNs in central nervous system.Secondly, through delivering TDNs and dsDNA to mouse hippocampus DG by mean of pressure microinjection,we fund that only TDNs could be uptaked by brain tissue; Then we studied tissue cells uptake in different periods of time, demonstrating it was 24 h that tissue could get best cell absorption efficiency;In the end,we improved cell absorption efficiency in brain tissue by in vivo electroporation and by adding surface active agent like 0.1% saponin and 0.2% Triton. For applying TDNs in mRNA imaging, we modified two florescence molecules in two edges of TDNs based on the FRET effect, and we ultimately confirmed that TDNs hold its structure integrity while it was in tissue for 24 h.Thirdly, according to the structure of TDNs, we redesigned the TDNs with an elongated DNA single strain on its one edge, which could binding to the mRNA probe. by screening the sequence of mRNA in brain tissue special cell, we finally generated the four mRNA detection models, which included actin-flare, vgat-flare, pv-flare and vip-flare. we first evaluated in vitro for their utility in quantifying complementary nucleic acid targets that had the same sequence as mRNA, it proved that four mRNA detection models could get the excellent signal-to-noise ratio and detection specificity. Vgat-flare could label the tissue cells well by mean of in vivo electroporation and by designing and assembling vgat-flare-cy5 for nerve cells labelling, we further proved the effectiveness of cells labeling with TDNs-flare.