Glutathione-Responsive Acyl-Modifications for Targeted RNA Decaging and Prolonged Protein Synthesis

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• Ribonucleic acid (RNA) modifications are crucial for enhancing RNA stability and function.
• Protecting the ribose 2'-hydroxy position is key for shielding RNA from degradation.
• Current RNA protection strategies face challenges in achieving controlled release and avoiding immune activation.

Purpose of the Study:

• To design and evaluate disulfide-based, self-immolative acyl conjugates for reversible RNA protection.
• To investigate the responsiveness of these modifications to endogenous glutathione levels.
• To assess the impact of these modifications on RNA stability, nuclease resistance, and protein production in cellular models.

Main Methods:

• Synthesis and characterization of novel disulfide-based acyl conjugates.
• Testing RNA recovery in solution and cultured cells using glutathione challenge.
• Evaluating nuclease resistance and cellular uptake of modified RNA.
• Measuring protein expression levels (green-fluorescent protein, nanoluciferase) in cell models over time.

Main Results:

• Designed acyl conjugates effectively protected the ribose 2'-hydroxy position of RNA.
• Modifications were responsive to glutathione, enabling efficient RNA recovery.
• Modified RNA demonstrated enhanced stability against nucleases and translational machinery.
• Significant increases in protein expression (up to 600%) and sustained production over 120 hours were observed compared to unmodified RNA.
• Minimal innate immune activation was noted.

Conclusions:

• Disulfide-based self-immolative conjugates offer a versatile strategy for reversible RNA protection.
• This approach enhances RNA stability, allows tunable release, and improves protein production.
• The findings support the development of next-generation RNA-based prodrugs with improved therapeutic profiles.