Recognition Of Sheared G:A Mismatched DNA By Ruthenium Polypyridyl Complex

The nucleic acid involved in the biological activities of lives, also closely related with cancer and other diseases, so DNA is usually used as a molecular target to study the pathogenesis of certain diseases. However, DNA base sequence doesn't set in stone. Generally, two mechanisms lead to DNA base sequence changes and thereby degrade the information content of DNA:1. The direct base mismatch may occur in the process of gene duplication. Although the high accuracy of DNA polymerase, there will still be a small part of mismatched base in the process of cell division and replication, moreover many mismatched base pairs exist in each new double helix;2. The formation of chemical modifications to polynucleotide caused by a constant barrage of damage, including redox events and alkylations. In addition, abusing of the drug may also damage DNA.If these damaged cells in the medium-term have not been completely killed, it will make DNA mutation, which may cause cancers and genetic diseases. Currently, the recognition and repair of mismatched systems has aroused widespread concern. Some studies show that the shear-type G:A mismatch is more difficult to repair than others in all of the mismatched base pairs. DNA containing G:A mismatch has wider minor groove than the normal B-DNA, moreover, nucleotides A and G slip out from the chains and form cross-linking accumulation with the complementary strand of nucleotides A and G. So DNA containing G:A mismatch is difficult to recognize and repair, and it has great research value and challenging. So far, the specific recognition of this mismatch has not been reported.Recently, our research group has studied interaction between racemic system and short-chain oligonucleotides by two-dimensional NMR, the result indicated that [Co(phen)2hpip]3+ can recognize sheared G:A mismatch preferentially and repair mismatched region. In view of this, we intend to design, synthesis and pick out recognition system for mismatched DNA, and to study the fine and exact modal of the interaction. Specific activities include the following:1. Picked out a ruthenium polypyridyl complex which can recognize the region adjacent to mismatches, [Ru(phen)2(DPPN)]2+ (DPPN=benzo [i] dipyrido [3,2-a:2',3'-c] phenazine), and have been ascertained its rough structure with infrared spectra, UV-visible absorption spectra, electrospray mass spectrometry, nuclear magnetic resonance spectra.2. We have studied the site selectivity of interaction between [Ru(phen)2(DPPN)]2+ and mismatched oligonucleotide d(CCGAATGAGG)2 and normal oligonucleotide d(CCTAATTAGG)2 by 1H,31P,2D NOESY and DQF-COSY spectra. The result displayed the interaction of the complex and mismatched DNA caused the resonances from DPPN and oligonucleotide broaden and the resonances from DPPN shift upfield. It indicated that intercalation is happened. There were many NOE cross-peaks in NOESY spectra. The result of more research indicated that DPPN ligand intercalates into the stacked bases at the mismatched adjacent regions A4A5:T6G7 from the major groove and C2G3:A8G9 from the minor groove. After the interaction of the complex and normal oligonucleotide, the resonances from DPPN broaden and shift upfiled, However, the sites of interaction couldn't be assigned as no NOE cross peaks in 2D NOESY spectrum.3. We studied the interaction between [Ru(phen)2(DPPN)]2+ and mismatched sequence as well as normal sequence DNA by theoretical molecular modeling. The results revealed that DPPN ligand intercalates into the stacked bases at the mismatched adjacent regions A4A5:T6G7 from the major groove. Moreover, the two isomers of complex could repair the sheared configuration when they interacted with mismatched DNA. The sheared conformation of mismatched DNA gradually changed to parallel form. The interaction mode for [Ru(phen)2(DPPN)]2+ and normal oligonucleotide is the intercalation of DPPN ligand to base stack in T6G7:A4A5 region from DNA minor groove, it reflects the groove selectivity and site specificity.4. The interaction of [Ru(phen)2(DPPN)]2+ with mismatched DNA was studied by UV spectra, fluorescence spectroscopy experiments. The complex shows fluorescence enhancement, absorption hypochromicity and red shift ascribe toπ-stacking between the great conjugate plane DPPN and DNA base pairs. The binding constant of complex and mismatched DNA is 7.20×105 M-1. After interact with normal oligonucleotide, the complex shows fluorescence enhancement, absorption hypochromicity and red shift, the binding constant of complex and normal DNA is 3.85×105 M-1.