ISSN:
1608-3245
Keywords:
catalytic RNA
;
ribozyme design
;
RNA synthesis
;
fluorescent oligonucleotides
;
reaction kinetics
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract We review our procedure for the rational design and the synthesis of oligoribonucleotides with particular reference to our recent studies of the development of a hairpin-derived twin ribozyme. Our work focuses on the design of ribozymes that are derived from naturally occurring structures but posses extended catalytic activities. Currently we are studying ribozymes that are capable of site-specific RNA double cleavage. We have designed these “twin ribozymes” by combining of two catalytic motifs in one molecule. In addition to kinetic analysis of the cleavage reaction, we are interested in the general phenomena of RNA folding and the characterization of RNA structure–function relationships. The developed twin ribozymes are particularly well suited for this purpose. They have to fold into a complex structure to achieve catalytic activity. Since there is a direct correlation between conformation and activity, the process of folding can be followed by monitoring the cleavage reaction. The twin ribozymes were synthesized by a combination of phosphoramidite chemistry and in vitro transcription techniques. We report the synthesis of a powerful reagent to replace tetrazole for activation of RNA phosphoramidites in automated solid-phase synthesis, allowing for coupling yields of 〉99%. Furthermore, we introduce a fast quantitative assay of ribozyme activity which is based on fluorescently labeled oligoribonucleotide substrates and DNA sequencer technology.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1026644313406
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