A structural analysis of in vitro catalytic activities of hammerhead ribozymes.
BACKGROUND
Ribozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes.


RESULTS
We employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and their catalytic activities were measured in vitro. We found that target disruption energy owing to the alteration of the local target structure necessary for ribozyme binding, and the total energy change of the ribozyme-target hybridization, are two significant parameters for prediction of ribozyme activity. Importantly, target disruption energy is the major contributor to the predictability of ribozyme activity by the total energy change. Furthermore, for a target-site specific ribozyme, incorrect folding of the catalytic core, or interactions involving the two binding arms and the end sequences of the catalytic core, can have detrimental effects on ribozyme activity.


CONCLUSIONS
The findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. Tools implementing these rules are available from the Sribo module and the Srna module of the Sfold program available through Web server at http://sfold.wadsworth.org.
Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA. yushao04@yahoo.com
Yu
Y
Shao
Susan
S
Wu
Chi Yu
CY
Chan
Jessie R
JR
Klapper
Erasmus
E
Schneider
Ye
Y
Ding
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing / Q000235:genetics; D002384:Catalysis; D017931:DNA Primers / Q000235:genetics; D005818:Genetic Engineering; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013816:Thermodynamics
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D009363:Neoplasm Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme