AUA-Cleaving Hammerhead Ribozymes: Attempted Selection for Improved Cleavage

Abstract

In order to find a ribozyme which can cleave the AUA triplet efficiently, the specificities and rates of intermolecular cleavage by the ribozyme of the satellite RNA of the barley yellow dwarf virus have been determined. Although it cleaves the AUA triplet in the plus strand of the viroid RNA, cleavage of AUC and AUU is more efficient, and AUG is essentially not cleaved. Attempts were made to increase cleavage efficiency by in vitro selection with randomization at positions 7, 10.1, and 11.1 in the core region. Fifteen clones were analyzed, two of which showed increased AUA cleavage efficiency. They have a G10.1.C11.1 base pair and a pyrimidine at position 7. This corresponds to the sequence of the consensus hammerhead ribozyme. Attempts to further increase cleavage efficiency by in vitro selection of the consensus hammerhead ribozyme with randomization of the 10 nucleotides in the core region or of the sBYDV ribozyme with 12 core nucleotides randomized were not successful.

Crystallization of RNA-protein complexes I. Methods for the large-scale preparation of RNA suitable for crystallographic studies

“…A possible remedy for the sequence restrictions imposed by the hammerhead ribozyme at the 3' cleavage site is the use of other self-cleaving sequences, such as the hairpin ribozyme at this position, the hairpin ribozyme has fewer sequence restrictions immediately 5' to the cleavage site and we show that it allows the synthesis of almost any sequence of RNA homogeneous in length. In addition, ribozymes with novel cleavage site specificities have been reported (Nakamaye & Eckstein, 1994), which may prove to be useful in the synthesis of RNAs terminating in different sequences.…”

Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

“…Recently, Nakamaye and Eckstein (46) reported on attempts to select for efficient AUA4-cleaving hammerhead ribozyme based on the catalytic domain of sBYDV. In the course ofthis study they have compared the relative cleavage rates of different AUX4 motifs in the same sequence context.…”

“…In our case the relative AUA4 cleavage rate was -36% of the AUC4 rate. As Nakamaye and Eckstein (46) 16.4 (3). It remains to be seen whether this is of any significance.…”

Direct selection for ribozyme cleavage activity in cells

“…In vitro selection has previously been used to evolve ribozymes (Wilson and Szostak 1999;Chen et al 2007) and even allosteric ribozymes (Breaker 2002). In particular, selection has previously been used to diversify the cleavage sites that can be utilized by the HHRz (Nakamaye and Eckstein 1994) and to optimize the sequence and function of the catalytic core (Tang and Breaker 1997), the stem-loop II (Long and Uhlenbeck 1994), or both (Ishizaka et al 1995).…”