Binding of HIV-1 TAR mRNA to a peptide nucleic acid oligomer and its conjugates with metal-ion-binding multidentate ligands

A peptide nucleic acid (PNA) oligomer and a series of PNA conjugates featuring covalently attached pendant 1,4,7,10-tetraazacyclododecane (cyclen) or bis((pyridin-2-yl)methyl)amine (DPA) moieties have been synthesized that are complementary to regions of the HIV-1 TAR messenger RNA stem-loop. Thermal denaturation studies, in conjunction win with native gel shift assays, suggest that the PNAs "invade'' TAR to produce a mixture of two 1: 1 PNA-TAR adducts, tentatively assigned as an "open-duplex'' structure, in which the TAR stem-loop dissociates and the PNA hybridizes with its RNA complement via Watson-Crick base-pairing, and a triplex-type structure, in which the initially displaced RNA segment is bound to the PNA: RNA duplex through Hoogsteen base-pairing. Thermal denaturation experiments with the TAR sequence and single-stranded RNA and DNA oligonucleotides, both in the presence and in the absence of Zn2+ ions, show that the introduction of cyclen or DPA ligand arms into the PNA oligomer leads to a small but reproducible increase in the T-m values. This is attributed to hydrogen-bonding and/or electrostatic interactions between protonated forms of cyclen/DPA and the cognate RNA or DNA oligonucleotide targets. Contrary to expectations, the addition of Zn2+ ions did not further enhance duplex formation through binding of Zn(II)-cyclen or Zn(II)-DPA moieties to the complementary RNA or DNA. Native gel shift assays further confirmed the stability increase of the metal-free cyclen-and DPA-modified PNA hybrids as compared with a control PNA sequence.