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Optimization and generality of a small deoxyribozyme that ligates RNA
Benjamin L Ricca1, Amanda C Wolf, Scott K Silverman
1Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Journal of Molecular Biology
|July 16, 2003
Summary
The 7Q10 deoxyribozyme efficiently ligates RNA, forming 2'-5' phosphodiester bonds. This optimized RNA ligase demonstrates broad substrate scope and practical utility for nucleic acid chemistry.
Area of Science:
- Biochemistry
- Molecular Biology
- Synthetic Biology
Background:
- Deoxyribozymes are catalytic DNA molecules with diverse enzymatic activities.
- The 7Q10 deoxyribozyme was previously identified for its RNA ligation capabilities.
- RNA ligation is crucial for various molecular biology applications.
Purpose of the Study:
- To optimize and assess the generality of the 7Q10 deoxyribozyme as a 2 ranscript-5' RNA ligase.
- To investigate the effects of mutagenesis on 7Q10's ligation activity and yield.
- To determine the substrate scope and optimal reaction conditions for 7Q10.
Main Methods:
- In vitro evolution was used to identify the parent 7Q10 deoxyribozyme.
- Comprehensive mutagenesis of the 7Q10 sequence was performed.
- RNA ligation assays were conducted under varying pH and temperature conditions.
Main Results:
- The parent 7Q10 sequence is optimal for RNA ligation yield.
- Mutations can increase ligation rate but decrease yield.
- 7Q10 ligates RNA substrates with the sequence motif UA GR, achieving >30% yield.
- Ligation is effective at pH 9.0 (1-2 hours) and pH 7.5 (12-24 hours).
- Significant ligation activity was observed for UA GY substrates.
Conclusions:
- The 7Q10 deoxyribozyme is a generally applicable and efficient 2 ranscript-5' RNA ligase.
- Its activity is dependent on substrate sequence and reaction conditions.
- 7Q10 offers practical utility and potential for mechanistic studies of nucleic acid catalysis.