A hammerhead ribozyme that was previously reported to have a rate of chemical cleavage 10-fold faster than that of conventional hammerheads was analyzed in greater detail. Although originally found as a bimolecular hammerhead assembled through helices I and II, fast cleavage was observed in hammerheads in the more conventional helix I-helix III form, provided the sequence of helix of the fast hammerhead was preserved. Mutations indicated that the fast rate of cleavage was due to the presence of both the U1.1-A2.1 and A1.2-U2.2 base pairs. The faster rate of cleavage was due to a small increase in the activation entropy of the reaction. In addition, we confirmed previous reports that increasing the length of helix by greater than five base pairs inhibits cleavage slightly and have uncovered a similar effect in helix II.
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