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A Pneumocystis carinii group I intron ribozyme that does not require 2' OH groups on its 5' exon mimic for binding to
S M Testa1, C G Haidaris, F Gigliotti
1Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Abstract:
The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37 degrees C, in 50 mM Hepes (25 mM Na+), 15 mM MgCl2, and 135 mM KCl at pH 7.5, the exogenous 5' exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2' OH groups of the 5' exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37 degrees C, the exogenous 5' exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2' OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.
Insights
Antisense compounds targeting fungal pathogen RNA offer new therapeutic strategies. Short oligonucleotides can bind tightly to structural RNAs by exploiting tertiary interactions, suggesting a novel approach for antifungal drug development.
Area of Science:
- Molecular Biology
- Antimicrobial Research
- RNA Therapeutics
Background:
- Opportunistic fungal infections are increasing in immunocompromised patients.
- Effective treatments are lacking, necessitating new therapeutic strategies.
- Antisense compounds targeting RNA are a promising approach.
Purpose of the Study:
- To develop a model system for antisense targeting of group I self-splicing introns in fungal pathogens.
- To characterize the binding of antisense agents to a fungal ribozyme.
Main Methods:
- Isolated and characterized the group I intron from Pneumocystis carinii.
- Constructed a catalytically active ribozyme (P-8/4x) for binding assays.
- Measured dissociation constants for exon mimic oligonucleotides using varying chemical compositions.
Main Results:
- The P-8/4x ribozyme showed high affinity for a 5' exon mimic (r(AUGACU)) due to tertiary interactions.
- Deoxynucleotide substitutions in the exon mimic enhanced binding, indicating 2' OH groups are not essential for stabilization.
- Oligonucleotides lacking 2' OH groups exhibited significantly tighter binding and specificity.
Conclusions:
- Short antisense oligonucleotides can effectively target structural RNAs by exploiting tertiary interactions.
- This suggests a new paradigm for antisense drug development, focusing on stabilizing tertiary structures.
- This approach holds potential for developing novel antifungal therapies.