Template-directed assembly and characterization of metallosalen-DNA conjugates

Nucleic acid template-directed synthesis represents a powerful method for the encoded synthesis of new bioconjugates. Our laboratory previously reported a strategy for the synthesis of a new metal-DNA hybrid, metallosalen-DNA, by the DNA or RNA template-directed cross-linking of two salicylaldehyde-modified DNA oligonucleotides. The current manuscript describes the optimal assembly requirements and biophysical characterization of metallosalen-DNA conjugates containing nickel and manganese ions. Competitive assembly reactions demonstrated the template-directed nature of metallosalen-DNA formation. A single metallosalen-DNA conjugate was assembled selectively in the presence of two pairs of salicylaldehyde precursor strands and a single DNA template. Assembly reactions were sensitive to base pair mismatches in the pairing arms. Single base mismatches resulted in a loss of metallosalen-DNA conjugate yield. Metallosalen-DNA assembly yields depended on the identity and length of the template spacer, the reaction pH, and the type of metal and diamine utilized in the assembly reaction. Metallosalen-DNA conjugates were stable to a variety of conditions, including extended incubation at 50 degrees C. Nickel metallosalen-DNA remained unchanged after incubation at 80 degrees C for 24 h, while decomposition of manganese metallosalen-DNA was observed under the same conditions. Circular dichroism (CD) spectroscopy indicated that DNA duplexes containing internal metallosalen moieties adopted B-form double helices. UV thermal denaturation analysis demonstrated that 32-nucleotide duplexes containing internal metallosalen modifications displayed melting temperatures approximately 5 degrees C less than unmodified DNA duplexes.