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 degrees 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'-termini of the two ODNs). No reaction was observed to occur either at 4 degrees C or at room temperature between pendent nucleotides separated by 9 nucleotides. Denaturing gel electrophoresis, MALDI-TOF MS and full nuclease digestion coupled with LC-MS were used to characterize the reaction products.