Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides

The ability of several DNA polymerases to catalyze the template-directed sy nthesis of duplex oligonucleotides containing a base pair between a nucleot ide with anhydrohexitol ring and its natural complement has been investigat ed. All DNA polymerases were able to accept the chemically synthesized anhy drohexitol triphosphate as substrate and to catalyze the incorporation of o ne anhydrohexitol nucleotide. However, only family B DNA polymerases succee ded in elongating the primer after the incorporation of an anhydrohexitol n ucleotide. In this family, Vent (exo(-)) DNA polymerase is the most success ful one and was therefore selected for further investigation. Results revea led that at high enzyme concentrations six hATPs could be incorporated; how ever, a selective incorporation proved only feasible under experimental con ditions where no more than two analogues could be inserted. Also the synthe sis of a mixed HNA-DNA sequence was examined. Kinetic parameters for incorp oration of one anhydrohexitol adenine nucleoside were similar to those of i ts natural analogue.