Thymine/uracil recognition in non-canonical nucleic acid structures by zinc macrocycle complexes
del Mundo, Imee Marie A.
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Zinc(II) complexes with and without pendant aromatic groups are recognition agents for single-stranded nucleic acids that contain thymine (T) or uracil (U). To develop these compounds further as nucleic acid binders, Zn(II)-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) complexes appended with a conjugated (4-methylquinoline, Zncy4q and 2-methylquinoline, Zncy2q ) and a non-conjugated (4-methylbiphenyl, Zncybp ) two-ring aromatic system were prepared. Binding affinities of these Zn(II) complexes to a series of hairpin oligonucleotides containing the strongly hairpin-nucleating 5'-GCA-3' or 5'-GAAA-3' loops with T/U in different secondary structures (duplex, loop, bulge, mismatch) was studied by using an ethidium bromide displacement assay and thermal denaturation studies. Results show that the parent Zn(II)-cyclen complex binds the oligonucleotides in the millimolar range. Binding with thymine bulge was also accompanied with an increase in thermal melting temperature (T m ), unlike the hairpins lacking a bulge which show destabilization. Zncy2q , with the nitrogen of the quinoline occupying a fifth coordination site, has the lowest affinity to all the motifs, with apparent dissociation constants ranging from 200–700 uM. Zncybp , presumably due to the internal flexibility of the two-phenyl rings relative to each other, binds mismatches and bulges with different affinities and modes. However, together with Zncy4q , both complexes have the low micromolar binding affinity for DNA sequences containing thymine as a single-base bulge, followed by hairpins with thymine in mismatched base pairs. Thermal melting studies show that Zncy4q's binding to TT and CT mismatches is accompanied by stabilizing interactions while Zncybp's interaction to TT and GT mismatches results in destabilization. Zncy4q was furthermore found to be > 100-fold selective for DNA containing a T-bulge over DNA containing G, C or A-bulges or hairpins lacking a bulge. Varying the flanking bases around the bulge revealed that Zncy4q prefers thymine bulges with purines as the bulge's flanking bases. Thermal denaturation and circular dichroism (CD) spectroscopy studies also show that Zncy4q bind to the thymine bulge without destablizing the stem of the oligonucleotide structure. This was later on confirmed from NMR imino proton studies. Further investigations using NMR spectroscopy showed large perturbations of the chemical shifts, especially that of the thymine bulge and the quinoline aromatic protons. Upfield chemical shifts were observed for most of the quinoline protons while downfield shifts were observed for the methylene (CH 2 ) linker of the complex as well as the T-Me, H6 and H1' of the thymine bulge. These changes led to our proposal that the thymine base and the quinoline group of the complex interacted by stacking, after the N3 deprotonated thymine coordinated with the Zn(II) metal center of the complex. Zn(II)-cyclen complexes with aromatic pendant groups, thus, represent a new class of bifunctional thymine-specific recognition agent. They constitute a richly versatile type of recognition agent for secondary structures containing non-canonical thymine or uracil nucleobases.