Preparation Of Satellite Assembly Probe And Antiviral Effects Of Chiral Cu_1.96s Nanoparticles

This paper is based on the synthesis of inorganic nanomaterials with upconverted luminescence signals and circular dichroic signals.By functionalizing the surface of these nanomaterials,two novel optically active inorganic nano-assembly probes are constructed.The multi-modal spectroscopic technology of nano-assembly probes are used to quantitatively analyze the mRNA and three metal ions in the live cells.In addition,the chiral copper sulfide nanoparticles that cleave the tobacco mosaic virus capsid protein(TMV CP)in a circularly polarized light-dependent and site-specifically manner are synthesized.The affinity effect and cleavage effect of chiral copper sulfide nanoparticles with different configurations to the target protein under natural light,left-handed circularly polarized light and right-handed circularly polarized light are studied.The molecular mechanisms of affinity and cleavage are explained.Finally,the prevention and treatment of virus infection at the plant level is realized.First,we design two of sulfhydryl DNA and modify the surfaces of AuNRs@Pt and UCNPs,respectively.AuNRs@Pt and UCNPs are assembled into satellite-like assemblies using a DNA sequence that specifically recognized target mRNA as a linker.In the assembly,the luminescence of UCNPs is effectively quenched due to the strong absorption of AuNRs@Pt in the range of 500 to 950 nm.When the target mRNA is present,the stronger binding capacity between the target mRNA and the linker DNA causes the UCNPs to fall off from AuNRs@Pt and restore their inherent luminescence.As the concentration of mRNA increases,the luminescence of the upconversion nanoparticles increases.By using assembly probes,we achieve specific detection of TK1 mRNA in living cells with detection range from1.17?65.21 fmol/10?g RNA,and the limit of detection of 0.67 fmol/10?g RNA.Importantly,the assembly probe has a wide range of versatility.Secondly,we develop a chiral assembly probe that can be specifically activated and simultaneously detected three metal ions in live cells.The chiral assembly is formed by the assembly of AuNRs@Pt dimer and UCNPs.In this assembly,the connecting DNA of the dimer is the Zn²⁺-specific DNAzyme and its protective DNA,and the substrate DNA is bound to the sequence outside the active center of the Zn²⁺-specific DNAzyme.Cu²⁺and Mg²⁺specific DNAzymes are integrated on a long strand of DNA,coupled onto the surface of AuNRs@Pt dimer.The substrate DNA of Cu²⁺is attached to the surface of UCNPs and is bound to the 3'end of the integrated DNAzyme through base complementary pairing.The substrate DNA of the Mg²⁺labeled with tetramethylrhodamine is bound to the 5'end of the integrated DNAzyme.The two protective DNAs are bound to the two active centers of the integrated DNAzyme.Due to the strong quenching effect of AuNRs@Pt dimer,the luminescence signal in the chiral assembly is quenched.The high efficiency photothermal conversion ability of the chiral assembly make it produce a significant thermal effect under the circularly polarized light of 980 nm,which cause the protective sequence to fall off.By precisely adjusting the light power,light time and the length of the substrate sequence,the substrate sequence keep bind.When the corresponding metal ion exists,it will bind to the active center of the enzyme,causing the DNAzyme to cut the corresponding substrate DNA so that produce the fluorescence of the dye and the luminescence of the UCNPs recovery.The different responsiveness of chiral assembly probes to circularly polarized light allows us to specifically activate the probe in the cell and avoid interference from extracellular targets.Therefore,the simultaneous quantitative detection of three metal ions in living cells are achieved,and standard curves for the detection of three metal ions in living cells are established.The limits of detection for Zn²⁺,Mg²⁺and Cu²⁺are 1.1 nM/10⁷cells,1.02 nM/10⁷cells and 0.45 nM/10⁷cells,respectively.Third,we synthesize chiral Cu_1.96S nanoparticles(chiral NPs).We discover the site-specific protein cleavage of chiral NPs induced by circularly polarized light.Negative staining-TEM,SDS-PAGE,circular dichroism(CD)spectroscopy,fluorescence spectroscopy,LC-MS and protein sequence analysis are used to characterize the cleavage reaction and find that the essence of the specific cleavage reaction is a hydrolysis reaction.The cleavage site is located between the Asp₁₀₁and Pro₁₀₂.The recognition protein sequence of the cleavage reaction is XAN(Q)PT(S)T(S)A,where X is any one of the 20 amino acids except Asp,and the third Asp can be Gln,Thr can be replaced by Ser.According to the sequence,the other three proteins are searched in the protein database and cut at the predicted site.Fourth,the isothermal titration calorimeter(ITC)test shows the affinity between D-Pen functionalized Cu_1.96S NPs(D-NPs)and TMV CP is 20 times higher than that of L-Pen functionalized Cu_1.96S NPs(L-NPs)and is 5,000 times higher than racemic Pen functionalized Cu_1.96S NPs(rac-NPs)as well as other ligands stabilized NPs.Molecular dynamics simulations reveal that the high affinity of D-NPs to TMV CP is due to the D-NPs interact with more inner polypeptide chains of TMV CP,forming more supramolecular interaction,and therefore more stably exists in the hole.Other ligand-stable NPs cannot enter the pore due to the weak affinity.D-NPs form a tighter hydrophobic binding pocket with the cleavage site comparing with L-NPs.The hydrophobic binding pocket result in a dihedral angle of 152°between the peptide bond of Asn₁₀₁residue and the Pro₁₀₂ring.The dihedral greatly affects the ability of the 1s orbital of the H atom on the CH₂unit to stabilize the?orbital.Therefore,D-NPs can weaken the peptide bond between the Asn₁₀₁and Pro₁₀₂.CPL can cause electrons to transfer from chiral NPs to the hole environment including the cleavage site.The negative potential surface formed by electron transfer of D-NPs is generally larger and overlaps with the cleavage site comparing with L-NPs.Thus,the electron on the N atom in Pro₁₀₂will increase sharply when formed a complex with D-NPs and exposed to CPL,making it vulnerable to the attack of H₂O molecules,thus causing the peptide bond to be hydrolyzed.In the end,chiral NPs are successfully used to effectively kill viruses in tobacco with high biocompatibility,which provide an effective antiviral method for the field of unclear plant antiviral.