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Alteration of hairpin ribozyme specificity utilizing PCR
P DeGrandis1, A Hampel, S Galasinski
1Department of Biological Science, Northern Illinois University, DeKalb 60115, USA.
PCR Methods and Applications
|December 1, 1994
Summary
Researchers can now easily modify trans hairpin ribozyme specificity using a novel PCR method. This technique rapidly generates new ribozyme templates for diverse applications, including antisense controls.
Area of Science:
- Molecular Biology
- Biochemistry
Background:
- Trans hairpin ribozymes are RNA molecules with catalytic activity.
- Altering ribozyme specificity is crucial for targeted gene manipulation and research.
Purpose of the Study:
- To develop a rapid and efficient method for altering the substrate specificity of trans hairpin ribozymes.
- To enable the generation of new ribozyme templates without synthesizing long oligonucleotides.
Main Methods:
- A Polymerase Chain Reaction (PCR) based method was employed.
- Two oligonucleotides and a target vector were used to create new ribozyme template sequences.
- Standard and modified ribozymes with altered specificity were generated and transcribed.
Main Results:
- The modified ribozymes demonstrated specific cleavage activity against a new substrate, beta-glucuronidase (GUS).
- The ribozymes lost activity against the original substrate, HIV-1 5' leader sequence.
- An inactive ribozyme variant was successfully produced for use as an antisense control.
Conclusions:
- This PCR-based method allows for quick and economical alteration of trans hairpin ribozyme specificity.
- The technique facilitates rapid assessment of ribozyme activity and development of targeted RNA tools.