Engineered pistol ribozymes selectively target KRAS G12V with enhanced efficacy by capping modification.

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Cancer Genomics

Background:

• The KRAS p.G12V mutation is a key driver in various cancers.
• Current RNA therapeutics like siRNA and ASO rely on sequence complementarity, limiting allele specificity.
• Pistol ribozymes offer unique structural recognition capabilities.

Purpose of the Study:

• To engineer a pistol ribozyme for selective targeting of the KRAS p.G12V mutation.
• To evaluate the specificity and efficiency of the engineered ribozyme against wild-type and mutant KRAS RNA.
• To investigate the impact of chemical modifications on ribozyme stability and therapeutic potential.

Main Methods:

• Rational engineering of a pistol ribozyme scaffold.
• Structure-based design utilizing pseudoknot-induced folding for substrate recognition.
• Allele-specific binding assays to differentiate KRAS wild-type and G12V mutant RNA.
• Comparative analysis of 5' end capping versus 3' end modifications for stability.

Main Results:

• The engineered pistol ribozyme demonstrated high specificity and efficiency in targeting the KRAS p.G12V mutation.
• The ribozyme's three-dimensional structure enabled precise discrimination between mutant and wild-type KRAS RNA.
• 5' end capping proved more effective than 3' end modifications for enhancing ribozyme stability and in vitro compatibility.

Conclusions:

• Engineered pistol ribozymes represent a promising class of therapeutic nucleic acids for KRAS mutation-driven cancers.
• Structure-guided, allele-specific RNA therapeutics offer a generalizable strategy for targeting oncogenic mutations.
• The findings highlight the potential of pistol ribozymes for precision oncology.