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 wer e obtained which cleave chimeric oligonucleotides at a single ribonucl eotide position embedded within a deoxyribonucleotide context in the p resence or absence of divalent metal ions. After several cycles of sel ection/amplification in the absence and in the presence of low amounts of Mg2+ two different types of catalysts emerged: one type depended s trongly 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 selec ted deoxyribozymes was carried out by chemical probing. The ribonucleo tide in the selected catalysts is unpaired and presents the cleavage s ite to the attacking nucleophile. Our results suggest that the main se lection criterion under metal-free conditions was a favourable arrange ment of the attacking nucleophile and the phosphate leaving group. The cleavage rates of the selected divalent metal independent catalysts a re within the same order of magnitude as the rate of metal independent substrate hydrolysis in the hammerhead ribozyme. One of the metal dep endent catalysts showed an unexpected preference for Ca2+ instead of M g2+. In this deoxyribozyme binding of Ca2+ occurred co-operatively whe reas binding of Mg2+ dib not. Comparison of the secondary structure an d reactivity of this catalyst with Mg2+ and Ca2+ suggests that here a special binding pocket for Ca2+ was selected. This deoxyribozyme achie ved a rate acceleration of substrate cleavage in the order of at least 10(4) compared to the uncatalysed reaction performing a cleavage mech anism similar to that of the hammerhead or hairpin ribozyme. (C) 1997 Academic Press Limited.