Sensing Systems For Transcription Factors Based On Nanomaterials And Their Biomedical Applications

Transcription factors?TFs?are DNA-binding proteins that play pivotal roles in cell development,differentiation and growth by regulating gene expression.Mounting evidence indicates that the dysregulation of TFs is involved in numerous pathological process.For example the excessive and persistent activation of nuclear factor kappa B?NF-?B?is implicated in autoimmune diseases,cancers,and viral infections.Another example is p53,whose abnormal expression is found in more than 50%of cancer cases,and its inactivation causes rapid growth of tumour.Therefore,TFs are not only important indicators in basic medical researches,but also potential diagnostic markers in clinical medicine.Therefore,it is meaningful to develop highly sensitive,efficient and low cost method for the detection of TFs.In this thesis,we engineered DNA-silver nanoclusters?DNA-AgNCs?,designed sensing strategies include enzyme-assisted signal amplification,nano-hybrid,and allosteric probes for the detection of TFs.The developed methods can not only be used for the quantification of target TFs,but also applied to the evaluation and screening of inhibitors.The following is the summarized content of each chapter:Chapter 1.Sensitive and Label-free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-assisted Signal AmplificationHerein,we firstly applied DNA-Ag nanoclusters molecular beacons?AgMBs?in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs.In the absence of TFs,a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe?referred as dsTFs probe?under exonuclease III?Exo III?digestion.Then,the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs,resulting in significant fluorescent decrease eventually.Conversely,the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state.To testify this rationale,we utilized nuclear factor-kappa B p50?NF-?B p50?as a model TFs.Owing to the amplification strategy,this method exhibited high sensitivity towards NF-?B p50 with a limit of detection of 10 pM,and a broad linear range from 30 pM to 1.5 nM.Furthermore,this method could be used to detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation.Finally,by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC₅₀of corresponding inhibitors.Chapter 2.DNA-Silver Nanoclusters/Polypyrrole Nanoparticles:A Label-Free and Enzyme-Free Platform for Multiplexed Transcription Factors DetectionIn this research,a hybrid material consisting of DNA-AgNCs and polypyrrole nanoparticles?PPyNPs?was built for TFs detection.The designed DNA-AgNCs have a hairpin-shaped nucleic acid architecture with a double-stranded stem for recognizing TFs and a single-stranded loop for interacting with PPyNPs.In the absence of TFs,DNA-AgNCs are absorbed to PPyNPs,resulting in fluorescent quenching.While in the presence of TFs,the binding between TFs and the DNA-AgNCs caused desorption of DNA-AgNCs via steric hindrance mechanism.Accordingly,the increase of fluorescence derived from desorption is used for quantifications.Derived from low non-specific protein absorption features of PyNPs,detection limit of 70 pM for NF-?B p50 and 110 pM for p53 were obtained.Then,by absorbing two kinds of DNA-AgNCs to PPyNPs,label-free multiplexed etection of TFs was first realized.Additionally,we suggested that this platform can be developed for drug screening by evaluating inhibitory effect of a pair of optical isomers towards TFs.Chapter 3.Detecting Transcription Factors with Allosteric DNA-Silver Nanocluster SwitchesIn this chapter,we report a DNA nanodevice,allosteric DNA-silver nanocluster switches?AgSwitches?,for TFs detection.The mechanism of this nanodevice is based on the binding-induced allostery whereby the binding between AgSwitches and TFs change the conformation of AgSwitches.The conformational change brings DNA-gNCs and guanine-rich enhancer sequences?GRS?into close proximity,generating fluorescent enhancement for quantifications.Our results revealed that the sequence design of AgSwitches can be rationally optimized according to stimulated free energy,and we demonstrated that this method can not only be used for detection of TFs in uclear extracts of cells,but also be developed as a screening platform for inhibitors of TFs.Overall,this work expanded the category allosteric DNA nanodevices by first introducing DNA-AgNCs into this area,and the obtained method was efficient for Fs-related investigations.