Applying DNA Rolling Circle Amplification In Fluorescence Imaging Of Cell Surface Glycans Labeled By Metabolic Method

Rolling circle amplification(RCA), an isothermal DNA replication technique, is a process for the enzymatic synthesis of long ssDNA with many repeating sequences.Metabolic labeling of glycans with chemical reporters(such as alkyne or azide) can incorporate chemical groups into growing cells' glycoconjugates, in conjunction with bioorthogonal chemistry, is a powerful tool for imaging glycome. Currently,metabolic labeling technology has been used for imaging cell surface glycan and glycoprotein. Here we report the development of a metabolic labeling- and RCA-based methodology that allows glycans imaging on cell surface.First, RCA was used for testing the cell surface glycans labeled by azido sugars.The advantage of this method is that it avoids introducing too much unnatural sugar,which can interfere with normal, physiological cell function. The optimum concentration of azide sugar used in this experiment was 5.0 ?M. Simultaneously, the enhanced fluorescence intensity conveniently facilitates the detection of cells' own biosynthetic glycans by simply using microplate reader. The results indicate that metabolically labelling ability is different for different carbohydrates and different cells. This phenomenon suggests that the involvement of azidosugar in glycan synthesis is not random and the glycan type is cell type dependent. Thus, using this method we can trace the variation in carbohydrate levels at different stages of the physiological process.Next, RCA was used for imaging the cell surface glycans labeled by mebolic method. The results showed that the amplification effect of DNA rolling circle amplification on metabolic labeling was very significant, a clearly fluorescence image can be get using low concentration of azido sugars.Finally, the RCA technique was adopted in a fluorescence resonance energy transfer(FRET)-based methodology that facilitated the glycan imaging of specific proteins on the cell surface. By combining with FRET, the glycosylation of specific glycocojugates can be detected using specific antibodies and proper azidosugars.This method is broadly applicable to imaging the glycosylation of cellular proteins,and is applicable to imaging glycosylation of a specific protein of interest.Our results highlight the applications of RCA in the metabolic glycan labeling,which can be used to monitor glycosylation status on cells and study the means by which glycosylation regulates cell function.