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 library of 5-glycosylated 2′-deoxyuridine triphosphates (dNTPs 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, longer side-chains of the oligosaccharide moiety considerably reduced the incorporation yield of nucleotides. On the other hand, the presence of the saccharides did not affect the serum stability of the 71-mer sequences containing the glyco-modified dNTPs, and were only slightly destabilizing duplex DNA. Interestingly, the presence of oligosaccharides highly increased the stability against the effect of the BfaI endonuclease. These findings show that 5-glycosylation with oligosaccharides expands the chemical alphabet of DNA without disrupting the base-pairing, offering a robust strategy for developing next-generation glyco-aptamers with enhanced endonuclease-stability and novel saccharides-mediated recognition capabilities.