Synthesis of 2′–modified nucleotides and their incorporation into hammerhead ribozymes

Beigelman, Leo; Karpeisky, Alexander; Matulić‐Adamić, Jasenka; Haeberli, Peter; Sweedler, David; Usman, Nassim

doi:10.1093/nar/23.21.4434

Cited by 55 publications

(30 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To obtain this product in the context of the hairpin ribozyme 13-mer substrate, it was necessary to produce a modified adenosine phosphoramidite for solid phase coupling [23]. …”

Section: Phosphoramidite Synthesismentioning

confidence: 99%

Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

Torelli

Spitale

Krucinska

et al. 2008

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

Reaction-intermediate analogs have been used to understand how phosphoryl-transfer enzymes promote catalysis. Herein we report the first structure of a small ribozyme crystallized with a 3′-OH, 2′,5′-linkage in lieu of the normal phosphodiester substrate. The new structure shares features of the reaction coordinate exhibited in prior ribozyme structures including a vanadate complex that mimicked the oxyphosphorane transition state. As such, the structure exhibits reaction-intermediate traits that allow direct comparison of stabilizing interactions to the 3′-OH, 2′,5′-linkage contributed by the RNA enzyme and its protein counterpart, ribonuclease. Clear similarities are observed between the respective structures including hydrogen bonds to the non-bridging oxygens of the scissilephosphate. Other commonalities include carefully poised water molecules that may alleviate charge build-up in the transition state and placement of a positive charge near the leaving group. The advantages of 2′,5′-linkages to investigate phosphoryl-transfer reactions are discussed, and argue for their expanded use in structural studies.

show abstract

“…To obtain this product in the context of the hairpin ribozyme 13-mer substrate, it was necessary to produce a modified adenosine phosphoramidite for solid phase coupling [23]. …”

Section: Phosphoramidite Synthesismentioning

confidence: 99%

Shared traits on the reaction coordinates of ribonuclease and an RNA enzyme

Torelli

Spitale

Krucinska

et al. 2008

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

show abstract

“…This notion was supported by a study where ribozymes were protected against 3'-exonuclease degradation by incorporation of a hairpin loop at the 3' end (Sioud et al, 1994). Alternatively, the 3' end can be protected by the incorporation of a thymidine in a 3'-3' link ("inverted T"), mimicking a hairpin structure while reducing the size of the ribozyme (Beigelman et al, 1995). Also, phosphorothioate linkages at the 5' end significantly enhance ribozyme stability over that of 2'-pyrimidine modifications and/or 3' phosphorothioate linkages alone .…”

Section: (I) Introductionmentioning

confidence: 97%

“…These effects are most likely produced by the strand-unfolding activity of this protein. Likewise, the RNA-binding heterogeneous nuclear ribonucleoprotein (hnRNP) Al has been successfully applied to increase ribozyme efficiency (Bertrand and Rossi, 1994 (Sioud and Jespersen, 1996) (Beigelman et al, 1995). Furthermore, similar globally modified ribozymes have been successfully applied in in vivo models (Lyngstadaas et al, , 1998Flory et al, 1996).…”

Section: (I) Introductionmentioning

confidence: 99%

Synthetic Hammerhead Ribozymes as Tools in Gene Expression

Lyngstadaas

2001

Critical Reviews in Oral Biology & Medicine

View full text Add to dashboard Cite

The assessment of genetic controls for sequential developmental processes such as tooth formation and biomineralization is often difficult in transgenic "knockout" models, where phenotypes reflect only the permanent eradication of a gene, and reveal little about the dynamic range of expression for the gene(s) involved. One promising strategy to overcome this problem is through the use of ribozymes, a class of metalloenzymes made entirely of ribonucleic acid (RNA), that are capable of cleaving other RNA molecules in a catalytic fashion. Their activity can be targeted against specific mRNAs by selection of unique sequences flanking a conserved catalytic motif. In synthetic ribozymes, specificity, stability, and cell permeability can be dramatically improved by the incorporation of chemically modified ribonucleotides. This review focuses on the design and application of hammerhead ribozymes, the best-known and most widely used class of RNA-based enzymes. So far, except for a few conserved structures at the catalytic core, no one particular model or superior ribozyme design has been identified. It may well be that each cell, tissue, and organism has different requirements for the uptake, activity, and stability of hammerhead ribozymes. However, designed ribozymes can be highly effective agents for timed and localized elimination of gene products. As the 3D structures of active hammerhead molecules are revealed, more effective ribozymes will be developed. Today, developments in ribozyme-mediated sequence-specific blocking of gene expression hold great promise for active RNA enzymes as tools in biomolecular research and for eliminating unwanted gene expression in human diseases.

show abstract

“…15,16 In order to further investigate the properties of oligonucleotides carrying these modi®cations we carried out preliminary attempts to explore the in¯uence of the 2 0 -methyl group on the orientation of the base as well on vicinal nucleotides. In view of the results afforded by these preliminary studies, and since differences between the hybridization af®nity of oligodeoxynucleotides carrying 2 0 -modi®ed moieties in certain positions and completely modi®ed sequences has been formerly observed 10 , we consider interesting to further investigate the synthesis and hybridization properties of oligonucleotides carrying 0 -C-methyl substituents with both arabino and ribo con®gurations in all but 3 0 -positions.…”

Section: Introductionmentioning

confidence: 99%