Thermodynamic dissection of the substrate-ribozyme interaction in the hammerhead ribozyme

The free energy of substrate binding to the hammerhead ribozyme was compare d for 10 different hammerheads that differed in the length and sequence of their substrate recognition helices. These hammerheads were selected becaus e neither ribozyme nor substrate oligonucleotide formed detectable alternat e secondary structures. The observed free energies of binding varied from - 8 to -24 kcal/mol and agreed very well with binding energies calculated fro m the nearest-neighbor free energies if a constant energetic penalty of Del ta G degrees(core) = +3.3 +/- 1 kcal/mol is used for the catalytic core. A set of substrates that contained a competing hairpin secondary structure sh owed weaker binding to the ribozyme by an amount consistent with the predic ted free energy for hairpin formation. These thermodynamic conclusions perm it the prediction of substrate binding affinities for ribozyme-substrate pa irs of any helix length and sequence, and thus, should be very valuable for the rational design of ribozymes directed toward gene inactivation.