The base-pairing properties of 5-mercuricytosine have been explored at the monomer level by NMR titrations and at the oligonucleotide level by melting temperature measurements. The NMR studies revealed a relatively high affinity for guanine, hypoxanthine, and uridine, that is, bases that are deprotonated upon coordination of Hg(II) . Within an oligonucleotide duplex, 5-mercuricytosine formed Hg(II) -mediated base pairs with thymine and guanine. In the former case, the duplex formed was as stable as the respective duplex comprising solely Watson-Crick base pairs. Based on detailed thermodynamic analysis of the melting curves, the stabilization by the Hg(II) -mediated base pairs may be attributed to a comparatively low entropic penalty of hybridization.
Nucleic acids that store and transfer biological information are polymeric diesters of phosphoric acid. Cleavage of the phosphodiester linkages by protein enzymes, nucleases, is one of the underlying biological processes. The remarkable catalytic efficiency of nucleases, together with the ability of ribonucleic acids to serve sometimes as nucleases, has made the cleavage of phosphodiesters a subject of intensive mechanistic studies. In addition to studies of nucleases by pH-rate dependency, X-ray crystallography, amino acid/nucleotide substitution and computational approaches, experimental and theoretical studies with small molecular model compounds still play a role. With small molecules, the importance of various elementary processes, such as proton transfer and metal ion binding, for stabilization of transition states may be elucidated and systematic variation of the basicity of the entering or departing nucleophile enables determination of the position of the transition state on the reaction coordinate. Such data is important on analyzing enzyme mechanisms based on synergistic participation of several catalytic entities. Many nucleases are metalloenzymes and small molecular models offer an excellent tool to construct models for their catalytic centers. The present review tends to be an up to date summary of what has been achieved by mechanistic studies with small molecular phosphodiesters.
An oligonucleotide incorporating a palladacyclic nucleobase has been prepared by ligand-directed metalation of a phenylpyridine moiety. This oligonucleotide hybridized with natural counterparts placing any of the canonical nucleobases opposite to the palladacyclic residue. The palladated duplexes had B-type conformation and melting temperatures comparable to those of respective unmodified duplexes with a single mismatch. In the duplexes placing C, G or T (but not A) opposite to the palladacyclic residue, greatly increased absorptivity suggested formation of a Pd -mediated base pair. Absorptivity and ellipticity of these duplexes persisted even at the highest temperatures applicable in T and CD experiments (90 °C). Evidently the Pd -mediated base pairs do not dissociate under the experimental conditions.
A 3-fluoro-6-methylaniline nucleoside was synthesized and incorporated into an oligonucleotide, and its ability to form mercury-mediated base pairs was studied.UV melting experiments revealed increased duplex stability with thymine, guanine, and cytosine opposite to the probe and a clear nucleobase-specific binding preference (T > G > C > A). Moreover, the 3-fluoro group was utilized as a spin label that showed distinct 19 F NMR resonance shifts depending on the complementary nucleobase, providing more detailed information on Hg(II)-mediated base pairing.
The capacity of three different purine bases, viz. 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, 2-(3,5-dimethylpyrazol-1-yl)adenine and 2,6-bis(2-acetyl-1-methylhydrazino)purine, to form metal-ion mediated base pairs with the native nucleobases has been examined. For this purpose, ribonucleosides derived from these bases were incorporated into an intrastrand or a 3'-terminal position of short 2'-O-methyl oligoribonucleotides and the hybridization properties of these base modified oligomers in the absence and presence of three different metal ions (Cu(2+), Zn(2+) and Pd(2+)) were studied by UV- and CD-spectrometry. The first two bases were found to stabilize short oligonucleotide duplexes when incorporated into the 3'-termini of both strands, even in the absence of divalent metal ions but especially in the presence of Cu(2+). The highest melting temperature determined for such a duplex was 71.8 °C, nearly 30 °C higher than the T(m) of the respective solely Watson-Crick paired duplex. Despite the dramatic stabilizing effect of the terminal metallo-base pairs, these short modified oligonucleotides retained sequence-selectivity for the internal Watson-Crick base pairs: two internal mismatches dropped the melting temperature to 10-11 °C. In an internal position, only 2,6-bis(3,5-dimethylpyrazol-1-yl)purine, which in the absence of metal ions was destabilizing, exhibited metal-ion-dependent stabilization of duplex formation with unmodified 2'-O-methyl oligoribonucleotides. The melting temperature in the presence of Cu(2+) was increased from 6 to 14 °C, depending on the identity of the opposite base.
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