Tetsuo Kozasa

HgII Ion Specifically Binds with T:T Mismatched Base Pair in Duplex DNA

Metal-mediated base pair formation, resulting from the interaction between metal ions and artificial bases in oligonucleotides, has been developed for its potential application in nanotechnology. We have recently found that the T:T mismatched base pair binds with Hg(II) ions to generate a novel metal-mediated base pair in duplex DNA. The thermal stability of the duplex with the T-Hg-T base pair was comparable to that of the corresponding T:A or A:T. The novel T-Hg-T base pair involving the natural base thymine is more convenient than the metal-mediated base pairs involving artificial bases due to the lack of time-consuming synthesis. Here, we examine the specificity and thermodynamic properties of the binding between Hg(II) ions and the T:T mismatched base pair. Only the melting temperature of the duplex with T:T and not of the perfectly matched or other mismatched base pairs was found to specifically increase in the presence of Hg(II) ions. Hg(II) specifically bound with the T:T mismatched base pair at a molar ratio of 1:1 with a binding constant of 10(6) M(-1), which is significantly higher than that for nonspecific metal ion-DNA interactions. Furthermore, the higher-order structure of the duplex was not significantly distorted by the Hg(II) ion binding. Our results support the idea that the T-Hg-T base pair could eventually lead to progress in potential applications of metal-mediated base pairs in nanotechnology.

Thermodynamic and structural properties of the specific binding between Ag+ ion and C:C mismatched base pair in duplex DNA to form C–Ag–C metal-mediated base pair

Metal ion-nucleic acid interactions have attracted considerable interest for their involvement in structure formation and catalytic activity of nucleic acids. Although interactions between metal ion and mismatched base pair duplex are important to understand mechanism of gene mutations related to heavy metal ions, they have not been well-characterized. We recently found that the Ag(+) ion stabilized a C:C mismatched base pair duplex DNA. A C-Ag-C metal-mediated base pair was supposed to be formed by the binding between the Ag(+) ion and the C:C mismatched base pair to stabilize the duplex. Here, we examined specificity, thermodynamics and structure of possible C-Ag-C metal-mediated base pair. UV melting indicated that only the duplex with the C:C mismatched base pair, and not of the duplexes with the perfectly matched and other mismatched base pairs, was specifically stabilized on adding the Ag(+) ion. Isothermal titration calorimetry demonstrated that the Ag(+) ion specifically bound with the C:C base pair at 1:1 molar ratio with a binding constant of 10(6) M(-1), which was significantly larger than those for nonspecific metal ion-DNA interactions. Electrospray ionization mass spectrometry also supported the specific 1:1 binding between the Ag(+) ion and the C:C base pair. Circular dichroism spectroscopy and NMR revealed that the Ag(+) ion may bind with the N3 positions of the C:C base pair without distorting the higher-order structure of the duplex. We conclude that the specific formation of C-Ag-C base pair with large binding affinity would provide a binding mode of metal ion-DNA interactions, similar to that of the previously reported T-Hg-T base pair. The C-Ag-C base pair may be useful not only for understanding of molecular mechanism of gene mutations related to heavy metal ions but also for wide variety of potential applications of metal-mediated base pairs in various fields, such as material, life and environmental sciences.

Thermodynamic Properties of the Specific Binding Between Ag+ Ions and C:C Mismatched Base Pairs in Duplex DNA

Metal-mediated base pairs formed by the interaction between metal ions and artificial bases in oligonucleotides have been developed for potential applications in nanotechnology. We recently found that a natural C:C mismatched base pair bound to an Ag+ ion to generate a novel metal-mediated base pair in duplex DNA. Preparation of the novel C-Ag-C base pair involving natural bases is more convenient than that of metal-mediated base pairs involving artificial bases because time-consuming base synthesis is not required. Here, we examined the thermodynamic properties of the binding between the Ag+ ion and each of single and double C:C mismatched base pair in duplex DNA by isothermal titration calorimetry. The Ag+ ion specifically bound to the C:C mismatched base pair at a 1:1 molar ratio with 106 M−1 binding constant, which was significantly larger than those for nonspecific metal ion–DNA interactions. The specific binding between the Ag+ ion and the single C:C mismatched base pair was mainly driven by the positive dehydration entropy change and the negative binding enthalpy change. In the interaction between the Ag+ ion and each of the consecutive and interrupted double C:C mismatched base pairs, stoichiometric binding at a 1:1 molar ratio was achieved in each step of the first and second Ag+ binding. The binding affinity for the second Ag+ binding was similar to that for the first Ag+ binding. Stoichiometric binding without interference and negative cooperativity may be favorable for aligning multiple Ag+ ions in duplex DNA for applications of the metal-mediated base pairs in nanotechnology.

The specific interaction between metal cation and mismatch base pair in duplex RNA

We have already found that mercury (II) cation specifically binds to T:T mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving T:T mismatch base pair by about 4 degrees C. We have also found that silver (I) cation specifically binds to C:C mismatch base pair in heteroduplex DNA, which increases the melting temperature of heteroduplex DNA involving C:C mismatch base pair by about 4 degrees C. In the present study, to examine whether the specific interaction between metal cation and mismatch base pair can be also formed in duplex RNA, we investigated the effect of the metal cation on the thermal stability of homoduplex and heteroduplex RNA. Addition of mercury (II) cation increased the melting temperature of heteroduplex RNA containing U:U mismatch base pair by about 6 degrees C. The thermal stability of homoduplex RNA containing U:A or A:U perfectly matched base pair and heteroduplex RNA containing A:A mismatch base pair was not significantly changed by the addition of mercury (II) cation. On the other hand, addition of silver (I) cation increased the melting temperature of heteroduplex RNA containing C:C mismatch base pair by about 4 degrees C. The thermal stability of homoduplex RNA containing C:G or G:C perfectly matched base pair and heteroduplex RNA containing G:G mismatch base pair was not significantly changed by the addition of silver (I) cation. We conclude that the specific interaction between metal cation and mismatch base pair can be formed in duplex RNA as in the case of duplex DNA.

Mismatch Base Pair Detection by Fluorescence Spectral Change Upon Addition of Metal Cation—Toward Efficient Analysis of Single Nucleotide Polymorphism

Addition of mercury (II) cation to fluorescent-labeled duplex involving a T:T mismatch base pair and silver (I) cation to fluorescent-labeled duplex involving a C:C mismatch base pair significantly changed the fluorescence intensity, but no significant change in the fluorescence intensity was observed for duplexes involving the other base pairs. The fluorescence spectral change upon addition of the metal cation can discriminate T:T and C:C mismatch base pairs from the other base pairs. Our results certainly support the idea that the fluorescence spectral change upon addition of the metal cation could be a convenient strategy for the mismatch base pair detection by the heteroduplex analysis, and may eventually lead to progress in single nucleotide polymorphism genotyping.