[Modified Oligonucleotides for Guiding RNA Cleavage Using Bacterial RNase P].

Area of Science:

• Molecular Biology
• Biochemistry
• Antimicrobial Research

Background:

• Bacterial ribonuclease P (RNase P) is a crucial enzyme for bacterial RNA processing.
• External guide sequences (EGS oligonucleotides) can direct RNase P to cleave specific RNA targets.
• Developing stable and effective EGS oligonucleotides for therapeutic applications is an ongoing challenge.

Purpose of the Study:

• To investigate the efficacy of novel modified external guide sequences (EGS oligonucleotides) in inducing target RNA hydrolysis mediated by bacterial ribonuclease P.
• To identify modifications that enhance EGS oligonucleotide stability in biological environments while preserving their RNA-cleaving activity.
• To explore the potential of these modified EGS oligonucleotides as novel antibacterial agents.

Main Methods:

• Synthesis and characterization of modified EGS oligonucleotides with alterations in the sugar-phosphate backbone.
• Assessment of the ability of modified EGS oligonucleotides to direct bacterial RNase P for targeted RNA cleavage.
• Evaluation of the stability of modified EGS oligonucleotides in simulated biological media.
• Identification of optimal modification patterns for enhanced stability and functional activity.

Main Results:

• Several modified EGS oligonucleotides demonstrated efficient hydrolysis of a model RNA target using bacterial RNase P.
• Modifications at the 2'-position (2'-O-methyl and 2'-fluoro) and internucleotide phosphates (phosphoryl guanidines) enhanced stability.
• Phosphoryl guanidine analogues of oligodeoxyribonucleotides showed stability in biological media and induced RNA hydrolysis.
• A balance between functional activity and stability was achieved with specific modifications.

Conclusions:

• Modified EGS oligonucleotides, particularly those with 2'-position or phosphoryl guanidine modifications, are effective in directing bacterial RNase P for targeted RNA cleavage.
• These modified EGS oligonucleotides exhibit enhanced stability in biological environments, making them promising candidates for therapeutic development.
• The findings highlight the potential of optimized EGS oligonucleotides as a novel class of antibacterial agents for targeted RNA degradation.