Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop...

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Catalysis

Background:

• The Neurospora VS ribozyme is a catalytic RNA known for specific substrate cleavage via a kissing-loop interaction (KLI).
• Engineering ribozymes allows for the development of novel biocatalysts with tailored functions.

Purpose of the Study:

• To explore the engineering potential of the VS ribozyme for cleaving alternative RNA substrates.
• To investigate the adaptability of the VS ribozyme's KLI for new interactions.

Main Methods:

• An innovative RNA engineering strategy combining rational design and combinatorial approaches was employed.
• Bioinformatic analysis of the Protein Data Bank identified structurally similar KLIs.
• Engineered substrate/ribozyme (S/R) pairs with alternative KLIs were kinetically and structurally characterized.

Main Results:

• Several engineered S/R pairs demonstrated substrate cleavage with significant catalytic efficiency.
• The catalytic activity of engineered pairs was generally reduced compared to the wild-type VS ribozyme.
• The study successfully adapted the VS ribozyme's KLI for interaction with alternative folded RNA substrates.

Conclusions:

• The kissing-loop interaction of the trans VS ribozyme is adaptable for cleaving diverse folded RNA substrates.
• This study presents a novel methodology for RNA engineering.
• The findings open avenues for designing customized ribozymes for specific RNA targets.