Template-directed synthesis and applications of metallosalen-DNA

Watson and Crick DNA base-pairing is central to many biological functions. It also facilitates chemical ligation of short DNA strands by bringing the reactive groups into proximity on a template. Utilizing these base-pairing interactions, the nucleic acid template-directed synthesis of metallosalen-DNA conjugates was developed. These conjugates introduce a single metal ion-ligand site into DNA, which demonstrates a number of practical uses.Two salicylaldehyde derivatives were designed, synthesized, and incorporated into a series of DNA strands. The SAL strand identities were confirmed by MALDI-TOF MS. The template-directed assembly of metallosalen-DNA conjugates was optimized for two metals, manganese and nickel. Assembly studies with different nucleoside template spacers indicated that Mn and Ni metallosalen-DNA conjugates form less efficiently with spacers other than (dT)2, and mismatched templates also lower conjugate yields. The identities of the metallosalen-DNA conjugates were verified by MALDI-TOF MS and base composition analysis. CD spectroscopy indicated that when in duplexes, the conjugates formed B-form helices. Thermal denaturation studies showed that metallosalen-DNA 32 nucleotide duplexes were &sim5°C less stable than unmodified duplexes. Nevertheless, nucleic acid template-directed molecular assembly offers a powerful approach for the addressable synthesis of new DNA conjugates in aqueous solution.Template-directed reactions also allowed hairpin metallosalen-DNA conjugate formation. Metallosalen-DNA hairpins could be assembled with or without metal ions. These new metal-DNA constructs were characterized by MALDI-TOF MS and base composition analysis. CD spectroscopy confirmed that the hairpin metallosalen-DNA conjugates adopt B-form DNA helices, and thermal denaturation studies indicated that the hairpin conjugates were more stable than natural DNA hairpins. A shorter Ni hairpin conjugate was evaluated for X-ray crystallography studies on the metallosalen-DNA conjugates.Nickel metallosalen-DNA conjugates have been applied to the oxidative site-specific cleavage of DNA. DNA sequencing reactions have shown that the Ni metal losalen-DNA conjugates cleave at guanine residues in a site-specific manner, providing oligonucleotide fragments terminated with phosphate groups. These oligonucleotide products have been confirmed with MALDI-TOF MS. Investigations of the cleavage mechanism indicated that a base-labile covalent adduct was formed between the Ni metallosalen-DNA and the target oligonucleotide.