Characterization of deoxy- and ribo-containing oligonucleotide substrates in the hammerhead self-cleavage reaction

Deoxynucleotides were introduced into a substrate fragment of the hammerhead RNA self-cleaving domain. A substrate lacking the 2′ hydroxyl adjacent to the cleavage site showed no detectable cleavage under a variety of reaction conditions. Competition experiments indicate that this fragment binds to the ribozyme with an affinity similar to the all RNA fragment, suggesting that the attacking 2′ hydroxyl does not substantially contribute to the binding of substrate to ribozyme. Similar competition experiments with the all DNA substrate indicate a much lower affinity for the ribozyme perhaps due to the lack of other 2′ hydroxyls. A substrate containing all deoxy residues except for a ribonucleotide at the cleavage site was also shown to be active.