Binding enhancement by tertiary interactions and suicide inhibition of a Candida albicans group I intron by...

Area of Science:

• Molecular Biology
• Antimicrobial Drug Discovery
• RNA Therapeutics

Background:

• Candida albicans is an opportunistic pathogen exhibiting increasing resistance to conventional antifungal treatments.
• RNA molecules, particularly catalytic RNAs, represent a viable class of targets for novel therapeutic interventions.
• Short oligonucleotides can be designed to bind RNA targets through Watson-Crick pairing and enhanced tertiary interactions.

Purpose of the Study:

• To investigate the potential of modified short oligonucleotides as therapeutics against Candida albicans by targeting its self-splicing group I intron in the LSU rRNA precursor.
• To design and evaluate hexanucleotide mimics of the 5' exon that incorporate backbone modifications for enhanced nuclease stability and binding affinity.
• To assess the inhibitory mechanism and efficacy of these oligonucleotides, particularly the ribophosphoramidate rn(GCCUC)rU, against catalytic RNA activity.

Main Methods:

• Design of hexanucleotide mimics based on the complex recognition elements of the 5' exon splice site of the C. albicans LSU rRNA group I intron.
• Modification of oligonucleotide backbones for increased stability against nucleases.
• Binding affinity assays comparing modified hexanucleotides to a mimic of the intron's internal guide sequence.
• Assays to evaluate the inhibition of precursor self-splicing and the mechanism of suicide inhibition.

Main Results:

• Modified hexanucleotides demonstrated significantly enhanced binding affinity (up to 100,000-fold) to a ribozyme derived from the intron compared to a control oligonucleotide.
• Several designed oligonucleotides effectively inhibited precursor self-splicing through a suicide inhibition mechanism.
• The ribophosphoramidate rn(GCCUC)rU exhibited potent inhibition, forming more trans-spliced than cis-spliced product at low concentrations (>100 nM) and 1 mM Mg(2+).

Conclusions:

• Short, nuclease-stable oligonucleotides can effectively target complex catalytic RNAs, such as the group I intron in C. albicans.
• The findings support the development of RNA-targeted therapeutics using modified oligonucleotides that exploit tertiary interaction elements for enhanced binding and inhibition.
• This strategy holds potential for combating drug-resistant pathogens like Candida albicans by targeting essential RNA functions.