ABSTRACT
2'-Modified pyrimidine nucleoside 5'-triphosphates comprising amino, imidazole and carboxylate functionality attached to the 5-position of the base were synthesized. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.
Subject(s)
Pyrimidine Nucleotides/chemical synthesis , RNA, Catalytic/chemistry , Drug Stability , Pyrimidine Nucleotides/chemistryABSTRACT
Dramatic improvements in ribozyme mimics have been achieved by employing the principles of small molecule catalysis to the design of macromolecular, biomimetic reagents. Ribozyme mimics derived from the ligand 2,9-dimethylphenanthroline (neocuproine) show at least 30-fold improvements in efficiency at sequence-specific RNA cleavage when compared with analogous o-phenanthroline- and terpyridine-derived reagents. The suppression of hydroxide-bridged dimers and the greater activation of coordinated water by Cu(II) neocuproine (compared with the o-phenanthroline and terpyridine complexes) better allow Cu(II) to reach its catalytic potential as a biomimetic RNA cleavage agent. This work demonstrates the direct mapping of molecular design principles from small-molecule cleavage to macromolecular cleavage events, generating enhanced biomimetic, sequence-specific RNA cleavage agents.
Subject(s)
Drug Design , Molecular Mimicry , Phenanthrolines/chemistry , Phenanthrolines/metabolism , RNA, Catalytic/metabolism , RNA/metabolism , Base Sequence , Catalysis , Cations, Divalent/metabolism , Copper/metabolism , Dimerization , Kinetics , Ligands , Molecular Structure , Phenanthrolines/chemical synthesis , Pyridines/metabolism , RNA/chemistry , RNA/genetics , RNA Probes/chemistry , RNA Probes/genetics , RNA Probes/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A series of novel 2'-modified nucleoside 5'-triphosphates was synthesized. The amino, imidazole, and carboxylate functionalities were attached to the 5-position of pyrimidine base of these molecules through alkynyl and alkyl spacers, respectively. Two different phosphorylation methods were used to optimize the yields of these highly modified triphosphates.
Subject(s)
Nucleotides/chemistry , RNA, Catalytic/chemistry , Nucleic Acid Conformation , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Previously developed '5-ribo' nuclease stabilized hammerhead motif was further refined by systematic incorporation of 1-(beta-D-xylofuranosyl) adenine (xA) and 1-(beta-D-xylofuranosyl) guanine (xG) in the place of conserved ribopurine residues of the catalytic core. Modified ribozymes substituted with xA at positions A15.1 and A6 demonstrated catalytic activity close to the parent stabilized ribozyme. Analogous guanosine substitutions at positions G5, G8, and G12 substantially lowered catalytic rates.
Subject(s)
Oligoribonucleotides/metabolism , RNA, Catalytic/chemistry , Adenine/analogs & derivatives , Catalysis , Chromatography, High Pressure Liquid , Guanine/analogs & derivatives , Models, Biological , Mutagenesis , Oligoribonucleotides/chemical synthesis , Organophosphorus Compounds/pharmacologyABSTRACT
We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups. The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described. DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex. The duplexes were treated with Escherichia coli RNase H. Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes. However, minor groove substituents inhibited RNA cleavage over a four-base region. Comparison was made with a DNA probe containing a 2'-OMe modification. Our results support enzyme binding in the minor groove of a DNA/RNA duplex. We do not observe cleavage directly across from the modified nucleoside. The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA. Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target. The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.
