Enzymatic Synthesis of Glyco-DNA Equipped with Oligosaccharides and Charged Monosaccharides

The therapeutic and biotechnological utility of synthetic oligonucleotides is constrained by the limited chemical diversity of canonical nucleotides and their susceptibility to nuclease degradation. Inspired by the hypermodified genomes of bacteriophages, where 5-glycosylated pyrimidines confer protection against host restriction enzymes, we report the design and synthesis of a scaffold of 5-glycosylated 2′-deoxyuridine triphosphates (dUTPs 1–7 ) bearing mono- (either neutral or charged moieties) and oligosaccharides affixed to position 5 of the nucleobase. The resulting glyco-modified nucleotides were well incorporated into DNA by different template-dependent polymerases, but much less by template-independent polymerases. While the presence of charged residues did not affect incorporation efficiency, glycan side-chain extension considerably reduced the incorporation yield of the glycosylated nucleotides. On the other hand, the saccharide appendages did not affect the serum stability of the 71-mer sequences containing the glyco-modified dUTPs and were only slightly destabilizing duplex DNA. Interestingly, the presence of short oligosaccharides highly increased the oligonucleotide stability against the BfaI endonuclease. These findings show that 5-glycosylation with short oligosaccharides could be a strategy to expand the chemical arsenal on nucleobases of DNA, offering a robust strategy for developing next-generation glyco-aptamers with enhanced endonuclease stability and novel saccharide-mediated recognition capabilities.