Chemoenzymatic Synthesis and Antibody Detection of DNA Glycoconjugates

A chemoenzymatic approach for the efficient synthesis of DNA−carbohydrate conjugates was developed and applied to an antibody-based strategy for the detection of DNA glycoconjugates. A phosphoramidite derivative of N-acetylglucosamine (GlcNAc) was synthesized and utilized to attach GlcNAc sugars to the 5‘-terminus of DNA oligonucleotides by solid-phase DNA synthesis. The resulting GlcNAc−DNA conjugates were used as substrates for glycosyl transferase enzymes to synthesize DNA glycoconjugates. Treatment of GlcNAc−DNA with β-1,4-galactosyl transferase (GalT) and UDP−Gal produced N-acetyllactosamine-modified DNA (LacNAc−DNA), which could be converted quantitatively to the trisaccharide Lewis X (LeX)−DNA conjugate by α-1,3-fucosyltransferase VI (FucT) and GDP−Fuc. The facile enzymatic synthesis of LeX−DNA from GlcNAc−DNA also was accomplished in a one-pot reaction by the combined action of GalT and FucT. The resulting glycoconjugates were characterized by gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and glycosidase digestion experiments. Covalent modification of the 5‘-terminus of DNA with carbohydrates did not interfere with the ability of DNA glycoconjugates to hybridize with complementary DNA, as indicated by UV thermal denaturation analysis. The trisaccharide DNA glycoconjugate, LeX−DNA, was detected by a dual DNA hybridization/monoclonal antibody (mAb) detection protocol (“Southwestern”):  membrane-immobilized LeX−DNA was visualized by Southern detection with a radiolabeled complementary DNA probe and by Western chemiluminescence detection with a mAb specific for the LeX antigen. The efficient chemoenzymatic synthesis of DNA glycoconjugates and the Southwestern detection protocol may facilitate the application of glycosylated DNA to cellular targeting and DNA glycoconjugate detection strategies.