Aptamer Recognition And HCR Signal Amplification Technology For Glycosylation Visualization Research

Malignant tumor is a disease caused by disordered physiological processes and excessive proliferation of normal cells.The cancer therapy and detection have still been a challenge,and early diagnosis can effectively improve the cure rate of patient.Therefore,the development of novel detection and diagnosis technology is one of the keys to advance the cancer research.Protein glycosylation,a very important posttranslational modification,is involved in various physiological activities and occurrence of diseases.Glycosylation is the transfer of carbohydrate chains to new synthesized peptide under the catalysis of glycosylation enzymes,which has an crucial impact on proteinic activity,structure and function.In recent years,some researchers argue that aberrant glycosylation is associated with the origin of malignant tumors,and this trend may have great potential for the early diagnosis.However,glycosylation analysis relies on development of glycoproteomic,mass spectrometry and microarray to analyze glycosylation.Thus,the visual mode for analyzing glycosylation need to be developed,which could provide a powerful tool to interpret the significance of glycosylation in cancer progress.We,combining the current research hotspots,based on the signal amplification strategy in DNA nanotechnology—Hybrid Chain Reaction(HCR)and Glucose Metabolic Oligosaccharide Engineering(MOE),and developed flexible,specific,and sensitive strategies for imaging cell surface protein-specific glycosylation.The detailed strategy is represented as follows,1.Intramolecular trigger remodeling-induced HCR for amplified detection of protein-specific glycosylationAccording to the characteristics of protein-specific glycosylation,we split DNA trigger of HCR into two parts,which are labeled to the target by protein recognition,and the glycosylation site through click chemistry,respectively.Subsequently,an activated trigger region of HCR can remodel through intramolecular proximity-induced DNA hybridization.We determined optimal experimental conditions by electrophoresis experiments and fluorescence experiments.The results show that this strategy has good selectivity and could distinguish between the glycosylation of CEM cells and the others cancer or normal cells.This method can significantly improve the contrast of imaging and has better selectivity.It will provide an effective method and tool for the analyzing glycosylation on cell membrane.2.DNA-displacement initiating HCR for Protein-specific Glycosylation imagingA glycoprotein usually contains multiple glycosylation sites.Using the previous strategy to analyze glycosylation can only use the labeled-probe at single site on the glycoprotein,which is not conducive to multi-site glycosylation detection.To solve this problem,we developed a localized strand replacement to trigger the HCR signal amplification method.By introducing a strand displacement reaction(DSA)on target.The aptamer-fused DNA probe APT targets glycoproteins,while the hairpin probe G1 is linked to the glycosylation site by click chemistry and hybridizes with APT to open the hairpin.Then the G2 hairpin probe hybridizes with the G1 hairpin to release the APT probe.The finally released APT probe hybridizes with the G1 probe at other sites,and the reaction is repeated until all glycosylation sites on the protein labeled with G1/G2.Then G2 fused with trigger could further initiate the HCR reaction.As result,each glycosylation site can form an HCR nanostructure.The results show that this method can be successfully triggered on the cancer cells and used to detect the glycosylation.This strategy may more accurately and reliably for reflecting the information of protein-specific glycosylation.