DNA sequences were isolated by in vitro selection for binding to N-methylmesoporphyrin IX (NMM), a molecule that behaves as a stable transition-state analogue for porphyrin chelatases. Clones approximately 280 and approximately 120 nucleotides long were obtained, which bound to NMM with sub-micromolar affinity but bound mesoporphyrin IX (MPIX), as well as various metalloderivatives of MPIX, with lower affinity. Footprinting experiments with dimethyl sulfate, DNase I, and bound hemin molecules activated by superoxide identified a series of short guanine-rich motifs to be the binding sites for the various porphyrins. One clone, PS2, examined in depth, gave a methylation footprint with bound NMM but not with bound MPIX nor with a number of metalloporphyrins. The binding domain PS2, synthesized as a short oligonucleotide, itself showed high-affinity binding to NMM. The binding sequences from different clones were loosely homologous, and the footprinting data were consistent with their folding to form one or more guanine quartets in the presence of NMM. Ultraviolet--visible absorption and circular dichroism spectroscopy of the DNA--NMM complexes indicates, however, that the interaction is not primarily intercalative in nature. The preferential binding of NMM by these aptamers raises the possibility of their being able to catalyze the chelation of metal ions by the porphyrin MPIX.

To guide the design of alternative genetic systems, we measured melting temperatures of DNA duplexes containing matched and mismatched nucleobase pairs from natural and unnatural structures. The pairs were analyzed in terms of structural features, including nucleobase size, number of hydrogen bonds formed, the presence of uncompensated hydrogen bonding functional groups, the nature of the bond joining the nucleobase to the sugar, and nucleobase charge. The results suggest that stability of nucleobase pairs correlates with the number of H-bonds, size complementarity, the presence of uncompensated functional groups, and the presence of charge on a nucleobase. Each of these properties appear to be more significant than the nature of the glycosidic bond and sequence context. The results provide guidelines for constructing stable Watson-Crick like nucleobase pairs with unnatural nucleobases. The experiments also demonstrate that expanded genetic systems can be constructed using size complementary nucleobase pairs that contain three hydrogen bonds.

Our results indicate that, although ribozymes are sometimes regarded generically to be metalloenzymes, the nucleic acid components of ribozymes may play a substantial role in the overall catalysis. Given that metal cofactors increase the rate of catalysis by ribozymes only approximately 10(2)-10(3)-fold above that of the DNAzyme described in this paper, it is conceivable that substrate positioning, transition-state stabilization or general acid/base catalysis by the nucleic acid components of ribozymes and DNAzymes may contribute significantly to their overall catalytic performance.

Antibiotic resistance arises from the maintenance of resistance mutations or genes acquired from the acquisition of adaptive de novo mutations or the transfer of resistance genes. Antibiotic resistance is acquired in response to antibiotic therapy by activating SOS-mediated DNA repair and mutagenesis and horizontal gene transfer pathways. Initiation of the SOS pathway promotes activation of RecA, inactivation of LexA repressor, and induction of SOS genes. Here, we have identified and characterized phthalocyanine tetrasulfonic acid RecA inhibitors that block antibiotic-induced activation of the SOS response. These inhibitors potentiate the activity of bactericidal antibiotics, including members of the quinolone, β-lactam, and aminoglycoside families in both Gram-negative and Gram-positive bacteria. They reduce the ability of bacteria to acquire antibiotic resistance mutations and to transfer mobile genetic elements conferring resistance. This study highlights the advantage of including RecA inhibitors in bactericidal antibiotic therapies and provides a new strategy for prolonging antibiotic shelf life.

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