Characterization and divalent metal-ion dependence of in vitro selected deoxyribozymes which cleave DNA/RNA chimeric oligonucleotides.
By in vitro selection, a variety of catalytic DNA oligonucleotides were obtained which cleave chimeric oligonucleotides at a single ribonucleotide position embedded within a deoxyribonucleotide context in the presence or absence of divalent metal ions. After several cycles of selection/amplification in the absence and in the presence of low amounts of Mg2+ two different types of catalysts emerged: one type depended strongly on Mg2+ or other divalent metal ions, the other type performed cleavage reactions independently of Mg2+ in the presence of spermine. Experimental analysis of the secondary structure of some of the selected deoxyribozymes was carried out by chemical probing. The ribonucleotide in the selected catalysts is unpaired and presents the cleavage site to the attacking nucleophile. Our results suggest that the main selection criterion under metal-free conditions was a favourable arrangement of the attacking nucleophile and the phosphate leaving group. The cleavage rates of the selected divalent metal independent catalysts are within the same order of magnitude as the rate of metal independent substrate hydrolysis in the hammerhead ribozyme. One of the metal dependent catalysts showed an unexpected preference for Ca2+ instead of Mg2+. In this deoxyribozyme binding of Ca2+ occurred co-operatively whereas binding of Mg2+ did not. Comparison of the secondary structure and reactivity of this catalyst with Mg2+ and Ca2+ suggests that here a special binding pocket for Ca2+ was selected. This deoxyribozyme achieved a rate acceleration of substrate cleavage in the order of at least 10(4) compared to the uncatalysed reaction performing a cleavage mechanism similar to that of the hammerhead or hairpin ribozyme.
Faulhammer
D
D
Institut für Biochemie-Genzentrum, München, Germany.
Famulok
M
M
D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis; D002413:Cations, Divalent / Q000494:pharmacology*; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded / Q000378:metabolism*; D019418:Evolution, Chemical; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000378:metabolism*; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
D002413:Cations, Divalent; D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D015335:Molecular Probes; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D009843:Oligoribonucleotides; D008274:Magnesium; D002118:Calcium