The Study Of The Interaction Between Pt-DNA And HMGB1 Based On Frame Nucleic Acid Platform

Cisplatin is usually used as an anticancer drug.Some studies have shown that the introduction of cisplatin can improve the cure rate from 5% to 90% in the process of combined chemotherapy for testicular germ cell tumors(TGCTs).Its anticancer mechanism lies in that after the platinum drugs are absorbed by cells,the platinum atoms and DNA Covalently combine to form intrachain and interchain crosslinks,forming Pt DNA adducts,causing DNA damage,thus causing a variety of cell reactions,such as replication block,transcriptional inhibition,blocking cell division cycle,and finally causing cancer cell death.In the process of DNA damage,there will be a series of response proteins,including HMGB1 protein,which is a high mobility group of chromosomal proteins and is considered to be the main binding protein of Pt DNA adducts.It has a very high affinity for Pt-DNA cross-linking.It can recognize and combine the damaged or curved DNA,shield the damaged sites,so it can inhibit the repair protein in cells to repair the damaged DNA,further promote the death of cancer cells,and achieve the purpose of cancer treatment.Therefore,it is of great significance to study the interaction between HMGB1 protein and Pt-DNA for understanding the pharmacological action of platinum drugs,DNA damage repair and drug resistance of cancer cells.At present,researchers have proposed different methods to study the interaction between Pt-DNA and HMGB1 protein,such as using X-ray diffraction to analyze crystal structure,molecular dynamics simulation,building DNA probe to capture interest protein for mass spectrometry analysis,etc.,but these methods still have disadvantages: on the one hand,they cannot carry out dynamic visualization research on the recognition and combination between the two,on the other hand,the experimental results As a result of the overall effect,some molecular information may be hidden in the population.So far,there is still a lack of a method to study the dynamic interaction between Pt-DNA and HMGB1 protein on a single molecular level.Because of the addressability of DNA nanostructures and it can be customized in size and shape,here,we build a rectangular DNA origami,and use it as a research platform for the interaction between Pt DNA and HMGB1 protein.Using singlemolecule observation technology,we can realize visual dynamic observation of the interaction between single molecules.The details are as follows:(1)Using gel migration assay(EMSA)and fluorescence resonance energy transfer(FRET)to characterize the interaction between HMGB1 protein and Pt-DNA at the macro level.Through EMSA,we can observe the difference between the two bands when they are combined and not combined.When the two bands are combined,the complex migration is slow,so the band position is high,and the phenomenon of tailing occurs.Fret test can further show that HMGB1 protein is a combination of damage sites,rather than other DNA sites.(2)Firstly,the DNA rectangular origami structure was constructed,and Pt-ssDNA was anchored on the origami platform as the research platform for the interaction between HMGB1 protein and Pt-dsDNA.AFM was used to observe the single molecular morphology of the products.It was found that when HMGB1 protein was identified and bound to the damage site,its height relative to the origami platform was about 1nm.(3)The interaction between HMGB1 protein and Pt-DNA was observed by TIRF.Firstly,a DNA rectangular origami structure with biotin modification was constructed and fixed on the imaging dish.After adding HMGB1 protein,the fluorescence signal intensity increased significantly after 500 s,indicating that HMGB1 protein and Pt DNA were combined,and the FRET signal was increased simultaneously,which further showed the binding of HMGB1 protein and damage site.Moreover,the co-location analysis of the two single molecule images shows that the co-location effect is good.Next,we performed single molecule imaging on the system without origami,and found that the co-localization effect and FRET efficiency of HMGB1 protein and Pt-DNA were lower.It is found that the binding efficiency of DNA origami as a research platform is 40%,30% higher than that of the system without origami,and the interaction between the two is balanced in about 100 s,while the traditional platform needs 500 s.These results show that DNA origami as a research platform can quickly identify the damage sites of HMGB1 protein,and the binding efficiency is higher.This method which takes DNA origami as the research platform realizes the visual research of HMGB1 protein and Pt-DNA interaction at the single molecule level by using the single molecule observation technology,avoiding the overall effect of the previous method,providing a new idea for the research of the interaction between the HMGB1 and Pt-DNA.And it is helpful to understand the mechanism of HMGB1 protein on DNA damage.