Self-assembled Multivalent DNA Nanostructures For Detection And Destruction Of Tumor Cells

Multivalent binding is defined as the simultaneous interaction of multiple binding groups on one molecule with the complementary receptors on another.In multivalent binding,the binding of the second ligand-acceptor can be considered as intramolecular interaction.In addition,multivalent binding can also be ascribed to the increased local concentration of ligand.Therefore,multivalent binding can induce higher affinity and selectivity compared to free ligands.Benefitting from the functionalized multivalent structure,high sensitive and selective probes or carriers for target delivery could be conveniently constructed,and be applied to chemical/biological information sensing,targeted drug delivery and immunotherapy.To realize multivalent binding,the self-assembly based strategy has attracted much attention because of the simple operation,mild condition and easy incorporation of multiple functional units.In this dissertation,we have developed a kind of multivalent DNA nanostructures,named as DNA nanocentipedes with controllable length and adjustable ligand valence.The nanocentipede was composed of a DNA scaffold based on hybridization chain reaction(HCR)and multiple ligands(including aptamers,molecular beacons and immunostimulatory oligonucleotides).The ligands were attached to the DNA scaffold through streptavidin-biotin affinity interaction.We focused on tumor cells as the objects of research to exploit the applications of the DNA nanocentipedes in assay,targeted drug delivery and immunotherapy.The research work of this dissertation included the following four parts:1.A high sensitive and selective assay for the human hepatoma cell line Bel-7404 was developed by using the multivalent DNA nanocentipede(Ls2n-Nces)based on aptamer Ls2n which was selected in our group.The Ls2n-Nces consisted of DNA scaffold and aptamer(Ls2n):(1)The DNA scaffold was obtained via HCR based on a trigger and two biotinylated DNA hairpins(H1 and H2);(2)Ls2n was labeled with biotin at 5’-end and fluorescent group at 3’-end,then the Ls2n was attached to the DNA scaffold through streptavidin-biotin affinity interaction.When target cell Bel-7404 existed,Ls2n-Nces bound with target cell rather than the control.Multivalent Ls2n(200)-Nces improved the selectivity and enhanced the signal intensity.Notably,the fluorescence signal of Ls2n(200)-Nces was enhanced significantly compared to that of free Ls2n.The probes have been successfully applied to detect single target cells.This work provided a novel strategy for highly sensitive cell detection,and the other tumor cells could be detected upon changing the related aptamers.2.To target the human hepatoma cell line SMMC-7721,a self-assembled multivalent DNA nanocentipede(Zyl-Nces)was construted by replacing the Ls2n with another aptamer Zyl which was also selected in our group.The Zyl-Nces demonstrated that higher valence could remarkably strengthen the binding to target SMMC-7721 cells.The Kd value of Zyl(200)-Nces decreased to 1/35 of that of free Zyl.Simultaneously,Zyl(200)-Nces could selectively recognize target SMMC-7721 and enhanced cellular internalization,while the selectivity was 4.6 times higher than that of free Zyl.In case of targeted drug delivery,the high drug loading capacity could reach to 500 Dox molecule per nanocentipede.Thus,this carrier provides an effective way to construct a versatile platform in diagnosis and therapy,once altering the aptamers to match different targets.3.In the third part,we developed an intracellular miRNA detection probe(MBF-Nces)by attaching fluorescent-labeled molecular beacons(MBFs)to the DNA scafford and then embedding quenchers(Doxorubicin,Dox)into the scaffold.Because the stem-loop(hairpin)of MBFs could drive the fluorescent dyes close to the quenchers,the fluorescent intensity was low("Off" state).When target miRNA was present,the MBF complemented with target miRNA,resulting in an open hairpin structure.The fluorescent groups were then away from quenchers,and turned to "On" state,i.e.high fluorescent intensity.The fluorescent intensity enhanced as the numbers of MBF on nanocentipede increased.For example,the fluorescent intensity of multivalent MBF(200)-Nces was 2.6 times to that of monovalent MBF(1)-Nces.In addition,MBF-Nces were also applied to detection of miRNA in living cells,in which the intracellular fluorescent intensity of MBF(200)-Nces was 2.1 times to that of MBF(1)-Nces.Compared to the previous method,our method processes several advantages,such as simple operation,high efficiency of cell internalization without any transfection reagents and quencher-free labeling.Importanly,the probe can detect various miRNAs by changing the MBF sequences.4.In order to expand the application of DNA nanocentipede for cancer therapy,we constructed a multivalent nanocentipede(CpG-Nces)by altering aptamer to biotinylated immunostimulatory oligonucleotide(CpG OND).CpG ODN could be specifically recognized by Toll-like receptor 9(TLR9)on the endosomal membranes of immune cells,and then stimulated secretion of a large amount of cytokines,thus destruction the tumor cells.Our studies showed that CpG-Nces could be internalized by RAW264.7 cells(mouse macrophage),and then induced large amount of cytokines secretion.Meanwhile,higher valence CpG-Nces could induce stronger immunostimulatory with higher secretion levels of cytokines in comparison with free CpG OND.When CCRF-CEM cells(Tlymphoblast leukemia,suspension cells)were co-cultured with RAW264.7 cells and CpG-Nces,higher valence numbers of CpG-Nces caused a lower percentage of living cells,indicating better immunotherapy,i.e.using multivalent CpG-Nces as an adjuvant for cancer treatment could significantly enhance the efficiency of immunotherapy.The above work suggests that the multivalent system has the potential to enhance the efficience of immunotherapy.