ArticleBMC biochemistry2005
Low-magnesium, trans-cleavage activity by type III, tertiary stabilized hammerhead ribozymes with stem 1 discontinuities.
Article in BMC biochemistry, 2005. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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16 citing papers in PubMed, 30 citations in OpenAlex.
- Enhanced hammerhead ribozyme turnover rates: Reevaluating therapeutic space for small catalytic RNAs.Molecular therapy. Nucleic acids · 2025Article
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- Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction.Biochemistry · 2015Article
- Efficient reverse genetics reveals genetic determinants of budding and fusogenic differences between Nipah and Hendra viruses and enables real-time monitoring of viral spread in small animal models of henipavirus infection.Journal of virology · 2015Article
- Bridging the gap between theory and experiment to derive a detailed understanding of hammerhead ribozyme catalysis.Progress in molecular biology and translational science · 2013Review
- Robust suppression of HIV replication by intracellularly expressed reverse transcriptase aptamers is independent of ribozyme processing.Molecular therapy : the journal of the American Society of Gene Therapy · 2012Article
- Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.Nucleic acids research · 2011Article
- Trans-cleaving hammerhead ribozymes with tertiary stabilizing motifs: in vitro and in vivo activity against a structured viroid RNA.Nucleic acids research · 2011Article
- Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity.Nucleic acids research · 2010Article
- Expanded hammerhead ribozymes containing addressable three-way junctions.RNA (New York, N.Y.) · 2009Article
- Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation.Journal of molecular biology · 2009Article
- Topological rearrangement yields structural stabilization and interhelical distance constraints in the Kin.46 self-phosphorylating ribozyme.RNA (New York, N.Y.) · 2006Article
- Leadzyme formed in vivo interferes with tobacco mosaic virus infection in Nicotiana tabacum.The FEBS journal · 2006Article
- Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads.Nucleic acids research · 2006Article
- Article
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2 authors at 1 institution in 1 country.
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Abstract
backgroundLow concentrations of free magnesium in the intracellular environment can present critical limitations for hammerhead ribozymes, especially for those that are designed for intermolecular (trans) cleavage of a host or pathogen RNA. Tertiary stabilizing motifs (TSM's) from natural and artificial ribozymes with a "type I" topology have been exploited to stabilize trans-cleaving hammerheads. Ribozymes with "type II" or "type III" topologies might seem incompatible with conversion to trans-cleavage designs, because opening the loop at the end of stem 1 or stem 2 to accommodate substrate binding is expected to disrupt the TSM and eliminate tertiary stabilization.
resultsStem 1, together with single-stranded segments capping or internal to this stem, contains both the substrate-binding and tertiary stabilization functions. This stem was made discontinuous within the sTRSV hammerhead ribozyme, thereby separating the two functions into discrete structural segments. The resulting ribozyme, designated "RzC," cleaved its 13 nucleotide target substrate at MgCl2 concentrations as low as 0.2 mM at 25 degrees C and 0.5 mM at 37 degrees C. Under multiple-turnover conditions, nearly thirty turnovers were observed at the highest substrate:RzC ribozyme ratios. Similar stabilization was observed for several derivatives of RzC. Catalytic activity was diminished or eliminated at sub-millimolar MgCl2 concentrations for ribozymes with weakened or deleted tertiary interactions. Eadie-Hofstee analysis revealed that the stabilized and non-stabilized ribozymes bind their substrates with equivalent affinities, suggesting that differences in observed activity are not the result of diminished binding. Some of the stabilized and non-stabilized ribozymes appear to fold into a heterogeneous collection of conformers, only a subset of which are catalytically active.
conclusionHammerhead ribozymes with the "type III" topology can be converted to a tertiary, trans-cleavage design. Separating the stabilization and substrate recognition functions of stem 1 increases cleavage activity at physiological concentrations of divalent magnesium while retaining recognition of exogenous targets. Trans-cleaving ribozymes that exploit the tertiary stabilizing motifs of all natural hammerhead topologies can therefore be used in intracellular applications.
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