Identification Of Binding Proteins By DNA-templated Chemistry And Photo-crosslinking Mass Spectrometry

Aptamers which were single-stranded DNA or RNA oligonucleotides can recognize diseased cells with the high selectivity,affinity and stability,showed great potential for applications in both diagnosis and therapy.It is well known that the apatmers can recognize proteins that are exposed on cell-surface to distinguish the types of cancer cells.However,it is still a big challenge to accurately identify the targets of aptamers due to the unstable,weak and noncovalent interactions between aptamers and their targets.Therefore the high sensitive analysis methods need urgently.Histone post-translational modifications(HPTMs)provide signal platforms to recruit protein or reader domains to regulate gene expression.In the process of these events,adjacent histone PTMs are able to altering the binding activity of readers toward their target marks.These covalent modifications can enhance,decrease,even eliminate the association of readers with chromatin.However,the “combinatorial readout” of PTMs are difficult to characterize and detect due to the lack of effective means of analysis on international.To solve above problems,we developed a new analysis method for the identification of the binding protein with high sensitivity.In our work,we used aptamer and peptide as molecules probes,respectively,based on the DNA-templated technology and a photo-crosslinking chemistry,and combined with mass spectrometry,aiming to develop a new analysis method for precise identification of target proteins.The main research contents are summarized as follows:Part one: By combining photo crosslinking method and DNA-templated chemistry,we have developed a dual-probe method for the detection of the aptamer-recognizing target protein.The probe can invert the weak and transient intermolecular interactions into covalent ones via the photo-affinity crosslinking reaction.Meanwhile,the crosslinking groups were conjugated to another single-stranded DNA skillfully,which otherwise might influent the interaction between aptamer and protein in some extent if the photo-reactive groups were conjugated in the middle position of aptamer which was too close to the binding site.This dual-probe strategy is more flexible and applicable,and has a separate and target-recognition independent probe that can effectively capture aptamer-target protein,which is a big advantage over those single-probe methods.Using lysozyme(LYS)as an example,the optimized probe showed high selectivity and specificity for the labeling and enrichment of aptamer-target protein in complex background.The method was further illustrated in egg white sample.Our work provides a novel tool for precise identification of proteins targeted by aptamers and shows a great potential in biomedical applications.Part two: On the basis of peptide recognition,DNA self-assembly and photo-crosslinking technology,a novel peptide probe was established for characterizing crosstalk between H3K4 tri-methylation(me3)and H3T3 phosphorylation(ph).Our results indicated that H3T3 phosphorylation inhibits the interaction between H3K4me3 and its reader,plant homeodomain(PHD)domain,which validated the literature reports.Our research shows that the new method has advantages on the analysis of the complex modified mediated protein-protein interactions and crosstalk between PTMs,could hold a potential application in biomedical fields.