Application Study Of Suzuki-Miyaura Coupling Based On BrdU In Detecting Cell Proliferation

Cell proliferation is an important biological process of the organism,and it has been frequently evaluated in a variety of biological research,such as anti-tumor drug screening,toxicity evaluation of organic compounds and nano-materials.DNA replication is a necessary step during cell proliferation,thus detection of DNA replication candirectly assess cell proliferation.In this type of cell proliferation assay,unnatural nucleosides were incorporated into the DNA of newly formed cells,then these unnatural nucleosides were recognized by various methods to reflect the status of cell growth.BrdU/antibody immunoaffinity,EdU/click chemistry,VdU/Inverse Diels-Alder reaction are the most commonly used methods for detecting DNA replication.BrdU is the least toxic unnatural nucleoside among them,but the detection relies on antibody immunoaffinity,which was limited by the poor specificity of commercial BrdU antibody.EdU and VdU are quite toxic to mammalian cells but the detection is based on the bioorthoganol reaction,which is fast,simple and specific.The Suzuki-Miyaura reaction is a cross-coupling reaction in which organic boron compounds coupled with organic halides or organic pseudo halides catalyzed by palladium or nickel.In recent years it has been widely used as an bioorthoganol reaction to modify and label cell components such as polysaccharides,proteins,polypeptides,oligonucleotides and DNA.In this study,we aim to label the BrdU which was incorporated into newly synthesized DNA during cell proliferation with Suzuki-Miyaura method to detect proliferating cells.We first evaluated the Suzuki-Miyaura reaction with BrdU and phenylboronic acid as the substrates at 70?.Next,we used different catalytic systems to optimize the reaction at 37?.The best experiment results were obtained while n-Bu4N+OH-was used as the base.Next we synthesized seven kinds of fluorescent boronic acid.Through multiple investigations at the chemical level,DNA level and cellular level,we found various factors interfere with cellular Suzuki-Miyaura labeling.After excluding these factors,we preliminary obtained the conditions for proliferating cells labeling.After re-optimized the Suzuki-Miyaura reaction of BrdU and phenylboronic acid,the reaction utilizing fluorescent boronic acid and BrdU as the substrates was performed and the result showed that the desired coupling product was generated.In the fixed cells,PdNP catalyzed cell labeling was investigated under four conditions,it was found that the best labeling results were obtained under hydrogen protection condition.In this method,oxidative addition between DTBPPS supported palladium nanoparticles and cellular BrdU were fulfilled under argon,then cells were labeled with fluorescent boronic under hydrogen.After that,we optimized the dye concentration,the dosage of palladium and the labeling time,finally we obtain the best condition for labeling proliferating cells.We validate the reliability of our approach using different methods.(1)HepG2,SH-SY5Y,MDA-MB-231,HeLa and A172 cells were labeled after increasing BrdU incubation,results indicated that the fluorescence intensity of Suzuki-Miyaura labeling was correlated to the concentration of BrdU.(2)Labeling experiments with increasing BrdU incubation time in HepG2 cells showed that the fluorescence intensity of Suzuki-Miyaura labeling was correlated to the amount of BrdU which incorporated into DNA.(3)Aphidicolin cell cycle arrest assay showed that the Suzuki-Miyaura method specifically labeled proliferating cells.(4)BrdU-EdU co-localization experiments showed that BrdU/Suzuki-Miyaura labeling and EdU/click-chemistry labeling were compatible with each other,and both of the two methods specifically labeled proliferating cells.Suzuki-Miyaura labeling method was used to detect proliferating cells in tumor tissues.The result show that it is easy to distinguish between proliferating cells and non-proliferating cells after Suzuki-Miyaura labeling.Combining our method with EdU/click chemistry to trace DNA synthesis using Pulse-Chase experiment showed that the proliferating cells were labeled monochromatic or two-color,and the two labels are spatial-temporal distinguishable.We used DEAC-boronic and FITC-boronic which emitting green fluorescence to label proliferating cells.The results show that two fluorescenct boronic specifically labeled proliferating cells,DEAC-boronic provide satisfied results while FITC-boronic gave high background fluorescent staining.In conclusion,we developed a method for the detection of cell proliferation using BrdU Suzuki-Miyaura labeling method.The method has the advantages of high specificity,high repeatability,low cost and compatibility with EdU.BrdU Suzuki-Miyaura labeling method circumvents the low specificity of BrdU antibody,and this brings wide applicability to BrdU cell proliferation assay.