2′,2′-Ligation demonstrates the thermal dependence of DNA-directed positional control

An oligodeoxynucleotide (ODN) containing a 2′-aminoalkylmercaptouridine was ligated chemically with another ODN containing a 2′-carboxyalkylmercaptouridine to produce a 2′,2′-amide linked conjugate. Templation by a DNA hairpin resulted in ligation yields of >95%. At room temperature, similar yields were obtained for amide ligations between pendent nucleotides located adjacent to each other or separated by three nucleotides. However, at 4 °C, ligation was vastly favored for the case where the pendent amine and carboxylate were attached to adjacent residues (i.e., at the 3′- and 5′-terminus of the two ODNs). No reaction was observed to occur either at 4 °C or at room temperature between pendent nucleotides separated by nine nucleotides. Denaturing gel electrophoresis, MALDI-TOF MS, and full nuclease digestion coupled with LC-MS were used to characterize the reaction products.