ACTIVITY DF THE HAMMERHEAD RIBOZYME UPON INVERSION OF THE STEREOCENTERS FOR THE GUANOSINE 2'-HYDROXYLS

Two guanosine 2'-hydroxyls in the hammerhead RNA complex at positions G5 and G8 are critical for efficient cleavage by this RNA catalyst. Th ese two functional groups are likely involved in the binding of the me tal cofactor, or they are involved in specific interresidue hydrogen-b onding interactions. The importance of the stereochemical positioning of both critical 2'-hydroxyls was investigated by comparing the cleava ge rates of three arabinosylguanine-substituted complexes (in which th e positions of specific guanosine 2'-hydroxyls were stereochemically a ltered by inverting the C2' stereocenter) with that of the native comp lex, as well as with the rates of the dG- and dFG-substituted complexe s fin which the 2'-hydroxyls are absent as the result of substitution by 2'-deoxyguanosine (dG) or 2'-deoxy-2'-fluoroguanosine (dFG)]. The G 5araG and G8araG complexes exhibit dramatically different cleavage rat es. The G5araG complex is essentially inactive, at least 10(5)-fold sl ower than the native complex. RNA cleavage by this analogue ribozyme i s also 1000-fold slower than cleavage by either the G5dG or the G5dFG ribozyme, both of which lack the 2'-hydroxyl at G5. By comparison, cat alytic efficiency of the G8araG complex as expressed by k(cat)/K-m is comparable with that of the native complex and some 2 orders of magnit ude more active than either the G8dG or the G8dFG complex.