Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme

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Abstract
Seven modified hammerhead ribozyme/substrate complexes have been prepared in which individual purine nitrogens, the guanine N7-, the guanine N2-, or the adenine N6-nitrogen, have been excised. The modified complexes were chemically synthesized with the substitution of a single 7-deazaguanosine (c7G), inosine (I), or nebularine (purine riboside, P) base analogue as appropriate for residues G5, G8, G12, A13, A14, or A15. Two of the base analogues, c7G5 and C7G8, occur in a 19-mer ribozyme, while the remaining three residues are present in a 24-mer substrate. Under stoichiometric conditions, four of the complexes, G5c7G, G8c7G, G12c7G, and A14P, are cleaved with relatively little change in rate when compared with the native complex. Two complexes, A13P and A15P, are cleaved some 6-8-fold slower than the native complex, while the G12I complex is reduced in rate by 50-fold. Steady-state kinetic analyses indicate that the cleavage efficiencies, as measured by kcat/KM values, for the G5c7G, G8c7G, and G12c7G complexes are only marginally reduced relative to the native complex. The values for the A13P, A14P, and A15P complexes are reduced by 25-, 15-, and 60-fold, respectively. These reductions in cleavage efficiency are primarily a result of lower kcat values. By comparison, the kcat/KM value for the G12I complex is decreased 450-fold relative to the native complex and is characterized by an 8-fold increase in KM and a kcat value that is reduced nearly 60-fold. These results indicate that the N2-amino group of G12 in the hammerhead ribozyme/substrate complex is critical for efficient cleavage activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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