Nanoparticle-based Ligand-nucleic Acid Macromolecular Interaction Fluorescence Analysis Method

Trans-activator of Transcription(Tat)and regulator of virion expression(Rev)proteins play a great role in the replication of Human Immunodeficiency Virus(HIV).Specifically,the alkaline area from Tat protein can specially bind to the bulge of trans-activating response(TAR)RNA,and arginine-riched peptide from Rev protein can also specially recognize the IIB stem-lopp of rev response element(RRE)RNA,which both matter to the viral replication.Therefore,ligand-RNA interaction assay and developing screening models would provide a strategy for inhibiting HIV replication.Some disadvantages existed when the conventional fluorescence-based methods were applied to the ligand-RNA interaction assay,so based on the fluorescent indicator displacement assay,and combined the advantages of nanotechnologis,some new methods were created for the small ligand-HIV-1 RNA interaction assay,and screening models for the antagonists of HIV peptide-RNA interaction were developed.Chapter 1 IntroductionIn sequence,the small ligand-biomacromolecule interactions,relevant nano materials and teconology,fluorescence-based methods and HIV peptide-RNA interactions were introduced.Finally,the research contents and objective were presented.Chapter 2 Probing interaction of a fluorescent ligand ICR 191 with TAR RNAIt’s difficult to find a small fluorescence indicator which possesses good fluorescence property,and can also block some RNA-targeted biological processes,and that’s why the fluorescent indicator displacement assay is hard to be applied into the study of ligand-RNA interaction.For the first time,a small fluorescence ligand,ICR 191,was found to interact with TAR RNA at the Tat binding site and compete with Tat.It was also observed that the fluorescence of ICR 191 could be quenched when binding to TAR RNA and recovered when discharged via competition with Tat peptide or a well-known Tat inhibitor,neomycin B.Mass spectrometry,circular dichroism and molecular docking were used to further confirm the interaction of ICR 191 with TAR RNA.Inspired by these discoveries,a primary fluorescence model for the discovery of Tat antagonists was built using ICR 191 as a fluorescence indicator and the screening feasibility of this model was evaluated in the cuvette.Finally,the feasibility of high throughout screening was evaluated in a format of 96-well plate.This ligand-RNA interaction could provide a new strategy for research aimed at discovering Tat antagonists.Chapter 3 Quantum dot-based ratiometric fluorescence assay for ICR 191-RRE RNA interactionSingle-wavelength measurements are usually carried out based on the fluorescence of indicator in the fluorescent indicator displacement assay,while compared with single-wavelength measurements,ratiometric fluorescence techniques have more advantages.A ratiometric fluorescence assay was applied in this chapter to monitor ligand-RNA interactions by using red-emitting CdTe quantum dots(QDs)coated with silica as a reference.A small fluorescence ligand,ICR 191,was found to interact with RRE at the Rev binding site and compete with the Rev peptide.After adding red-emitting QDs to the interaction system,it was observed that ICR 191 did not fluoresce upon the addition of RRE,and fluorescence recovered when ICR 191 was displaced by a Rev model peptide,whereas the fluorescence of QDs remained constant.Furthermore,variations in the fluorescence ratios between ICR 191 and QDs were exploited to characterize the interactions of Rev with two known antagonists by using RRE RNA with ICR 191 as a fluorescence indicator.The advantages of ratiometric fluorescence were taken to the ligand-RNA interaction assay,and it can potentially be developed to build a screening model for assessing antagonists of the Rev binding element in RRE.Chapter 4 Fluorescent DNA-protected silver nanoclusters for ligand-HIV RNA interaction assayMitoxantrone(MTX)dose not emit fluorescence itself,so it’s not available to study MTX-HIV RNA interaction based on its own fluorescence.DNA-protected silver nanoclusters(AgNCs)were used to investigate ligand-RNA interactions for the first time in this chapter.MTX was found to quench the fluorescence of AgNCs.After adding HIV TAR RNA or RRE RNA to AgNCs-MTX mixtures,the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs.The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations.The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored.This study founded a novel ligand-RNA interaction model,and further provides a new strategy for the interactions between non-fluorescent molecule and HIV RNAs indirectly using the fluorescence method.Chapter 5 Graphene oxide-based and proflavine-indicated fluorescence polarization assay for the discovery of Rev antagonistsCompared with fluorescence spectroscopy,fluorescence polarization(FP)possesses anti-environment disturbance abilitis.However,as a traditional method for the ligand-RNA interaction assay,fluorescence polarization usually has the nonsignificant changes in polarization signal.It was reported that proflavine could recognise HIV RRE RNA,thus inhibiting its interaction with Rev peptide.Owing to the non-significant difference in fluorescence polarization between the proflavine and proflavine-RRE complex,it is not practicable for theidentification of antagonists of HIV Revpeptideusing proflavineas a FP indicator.In this chapter,graphene oxide(GO)was introduced to amplify the FP signal to enhance the sensitivity for the identification of Rev antagonists,thereby enabling the development of a primary GO-based FP model for the discovery of Rev antagonists.The effect of the amount of GO on the FP changes was investigated via displacement assay using a known Rev antagonist,neomycin B,and the competition between Rev and proflavine was verified.This primary model was further evaluated using other test drugs.The mechanism underlying the changes in FP caused by the addition of neomycin B in the presence of GO was further explored.This model can potentially provide a new screening strategy for assessing antagonists of the Rev binding element in RRE.Chapter 6 Fluorescent MnO2 nanosheet-assisted ligand-DNA interaction-based fluorescence polarization biosensor for the detection of Ag+ ionsSilver ions(Ag+)are highly toxic to aquaticorganisms and accumulate in the human body via the food chain.Therefore,the development of sensitive and selective quantitative analytical methods for detecting trace amounts of these ions is necessary.For the first time based on the role of Mn02 nanosheets in enhancing the fluorescence polarization,a label-free MnO2 nanosheet-assisted,ligand-DNA interaction and fluorescence polarization-basedmethod was developed,for the first time,for sensitive detection of Ag+.The addition of Ag+ to the preformed proflavine-DNA complex induced the release of proflavine,which elicited weak changes in fluorescence polarization.The subsequent addition of MnO2 nanosheets magnified the observed changes,making this a feasible method for the detection of Ag+.The calibration graphs indicated good linearity over the concentration ranges of 30-240 nM for Ag+,with a detection limit(S/N = 3)of 9.1 nM.This method additionally exhibits high selectivity.The mechanism underlying the changes in fluorescence polarization caused by the addition of Ag+in the presence of MnO2 nanosheets was further explored.Chapter 7 Small ligand-DNA Interaction Assay based on Confocal Fluorescence Correlation SpectroscopySignificant fluorescence signal changes can guarantee characterization of small ligand-DNA using the fluorescent spectrometry.However,this method would become unavailable without significant signal changes.We first chose rhodamine B(Rb)and herring sperm DNA as model,to investigate the feasibility of using confocal fluorescence correlation spectroscopy(FCS)to characterize small ligand-DNA interaction,and then applied this method to investigate the binding of a novel unimolecular submersible nanomachine(USN)to DNA.This was the first study to study ligand-DNA interaction based on FCS,and would provide a new strategy for assessing other ligand-micromolecular interactions.Chapter 8 ConclusionSummarize the whole thesis,and then point out the shortages of the whole sdudy.