Selection of the Most Potent Specific On/Off Adaptor-Hepatitis Delta Virus Ribozymes for Use in Gene Targeting

Lévesque, Michel V.; Rouleau, Samuel G.; Perreault, Jean‐Pierre

doi:10.1089/nat.2011.0301

Cited by 6 publications

(5 citation statements)

References 36 publications

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“…Before our discovery of a long J1/2 insert region in the mosquito ribozymes, a structured J1/2 was shown to stabilize the P2 helix in an engineered trans -ribozyme with an additional 7 base-pair stem (P1.2-like) in the J1/2 region 36 . The J1/2 region was also used to design a specific on/off adaptor system for a trans HDV ribozyme, which acts as a switch with the ribozyme “on” only when an allosteric substrate is present 37 . Moreover, using footprinting and FRET assays, the J1/2 region was implicated as the critical element that determines the different structural characteristics between the cis - and trans -acting HDV ribozymes 20 .…”

Section: Discussionmentioning

confidence: 99%

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

Webb

Nguyen

Myszka

et al. 2016

Sci Rep

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Self-cleaving ribozymes fold into intricate structures, which orient active site groups into catalytically competent conformations. Most ribozyme families have distinct catalytic cores stabilized by tertiary interactions between domains peripheral to those cores. We show that large hepatitis delta virus (HDV)-like ribozymes are activated by peripheral domains that bring two helical segments, P1 and P2, into proximity – a “pinch” that results in rate acceleration by almost three orders of magnitude. Kinetic analysis of ribozymes with systematically altered length and stability of the peripheral domain revealed that about one third of its free energy of formation is used to lower an activation energy barrier, likely related to a rate-limiting conformational change leading to the pre-catalytic state. These findings provide a quantitative view of enzyme regulation by peripheral domains and may shed light on the energetics of allosteric regulation.

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Section: Discussionmentioning

confidence: 99%

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

Webb

Nguyen

Myszka

et al. 2016

Sci Rep

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“…For the long substrate presented here (Figure A), the overall specificity is almost 10 11 (for the 6 and 12 bp of P1 and P1.2, respectively, 4 6 × 4 12 = 6.9 × 10 10 ), much greater than the theoretical sequence diversity of any genome (e.g., the length of the haploid human genome is ∼3 × 10 9 bp, and its theoretical sequence diversity is ∼6 × 10 9 when the genome is transcribed in both directions). This result implies that the Agam-2-1-like trans -cleaving ribozymes could have evolved (or be engineered) to cut unique target RNAs even in organisms with highly complex transcriptomes. Motivated by this hypothesis, we designed a bioinformatics approach to search for potential ribozyme–substrate pairs in the A. gambiae genome.…”

Section: Resultsmentioning

confidence: 99%

“…The trans -HDV ribozyme was previously constructed to study the kinetics of cleavage , and for gene knockdown experiments; ,− however, the short 7 nt binding region for the trans -ribozyme substrate provided little utility for the construct, even though it was useful for defining the kinetic parameters of the HDV ribozymes. The long P1.2 insertion discovered in A. gambiae and other insect ribozymes ,, could serve for stronger target RNA recognition, if the domain was bisected to yield two RNAs, a ribozyme and a long substrate, and a similar approach has been proposed for engineering HDV ribozymes into more specific trans cleavers. , To evaluate the influence of the long target binding sequence, it is imperative that the kinetic parameters of such ribozymes be measured to assess the usefulness of these catalysts for cellular targeting and cleavage of single-stranded RNAs. We therefore set out to study the mechanism of cleavage by utilizing the intermolecular HDV-like ribozyme and its substrate derived from the drz-Agam-2-1 ribozyme.…”

Section: Discussionmentioning

confidence: 99%

“…75 The calculated free energies and predicted base pairs of the RNA heterodimers are presented in the Supporting Information. ribozymes could have evolved (or be engineered 60 ) to cut unique target RNAs even in organisms with highly complex transcriptomes. Motivated by this hypothesis, we designed a bioinformatics approach to search for potential ribozyme− substrate pairs in the A. gambiae genome.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

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Kinetic Parameters of trans Scission by Extended HDV-like Ribozymes and the Prospect for the Discovery of Genomic trans-Cleaving RNAs

Webb

Lupták

2018

Biochemistry

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Hepatitis delta virus (HDV)-like ribozymes are self-cleaving catalytic RNAs with a widespread distribution in nature and biological roles ranging from self-scission during rolling-circle replication in viroids to co-transcriptional processing of eukaryotic retrotransposons, among others. The ribozymes fold into a double pseudoknot with a common catalytic core motif and highly variable peripheral domains. Like other self-cleaving ribozymes, HDV-like ribozymes can be converted into trans-acting catalytic RNAs by bisecting the self-cleaving variants at non-essential loops. Here we explore the trans-cleaving activity of ribozymes derived from the largest examples of the ribozymes (drz-Agam-2 family), which contain an extended domain between the substrate strand and the rest of the RNA. When this peripheral domain is bisected at its distal end, the substrate strand is recognized through two helices, rather than just one 7 bp helix common among the HDV ribozymes, resulting in stronger binding and increased sequence specificity. Kinetic characterization of the extended trans-cleaving ribozyme revealed an efficient trans-cleaving system with a surprisingly high K', supporting a model that includes a recently proposed activation barrier related to the assembly of the catalytically competent ribozyme. The ribozymes also exhibit a very long k for the products (∼2 weeks), resulting in a trade-off between sequence specificity and turnover. Finally, structure-based searches for the catalytic cores of these ribozymes in the genome of the mosquito Anopheles gambiae, combined with sequence searches for their putative substrates, revealed two potential ribozyme-substrate pairs that may represent examples of natural trans-cleaving ribozymes.

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“…While the half-life of HDV-Rzs is superior to other RNA molecules, owing in part to its formation of a highly stable secondary structure (57), this stability may be improved by chemical modifications as has been done for siRNA. However, these alterations can decrease their effectiveness, and in the case of SOFA HDV-Rzs, may not offer much advantage (63,64).…”

Section: Discussionmentioning

confidence: 99%

Effective Suppression of Chikungunya Virus Infection in Cell Cultures using Hepatitis Delta Ribozymes

Fraser,

Kucharski,

Loh

et al. 2024

Preprint

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Chikungunya virus (CHIKV) is an emerging pathogen with widespread distribution in regions of Africa, India, and Asia that threatens to spread into temperate climates following the introduction of its major vector, Ae. albopictus. Recent cases have been documented in Europe, the Caribbean, and the Americas. CHIKV causes a disease frequently misdiagnosed as Dengue fever, with potentially life-threatening symptoms that can result in long term debilitating arthritis. There have been ongoing investigations of possible therapeutic interventions for both acute and chronic symptoms, but to date none have proven effective in reducing the severity or lasting effects of CHIKV disease. Recently, a promising vaccine candidate has received accelerated FDA approval, indicating the importance of remedies to this emerging worldwide health threat. Nonetheless, therapeutic interventions for CHIKV and other mosquito borne virus diseases are urgently needed yet remain elusive. The increasing risk of spread from endemic regions via human travel and commerce, coupled with the absence of a vaccine or FDA approved therapeutic, puts a significant proportion of the world population at risk for this disease. In this report we explore the possibility of using SOFA (Specific On/oFf Adapter) Hepatitis Delta Virus Ribozymes (HDV-Rzs) as antivirals in cells infected with CHIKV. The results we obtained suggest there could be some role in using these SOFA HDV molecules as antiviral therapies for not only CHIKV, but potentially other viruses as well.

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Selection of the Most Potent Specific On/Off Adaptor-Hepatitis Delta Virus Ribozymes for Use in Gene Targeting

Cited by 6 publications

References 36 publications

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes

Kinetic Parameters of trans Scission by Extended HDV-like Ribozymes and the Prospect for the Discovery of Genomic trans-Cleaving RNAs

Effective Suppression of Chikungunya Virus Infection in Cell Cultures using Hepatitis Delta Ribozymes

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