Deoxyribozymes with 2'-5' RNA ligase activity

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Deoxyribozymes, or DNA enzymes, offer catalytic functions traditionally associated with RNA.
• Efficient methods for creating specific RNA linkages are crucial for RNA engineering and structural studies.

Purpose of the Study:

• To identify and characterize novel deoxyribozymes capable of ligating two RNA substrates.
• To assess the impact of the newly formed 2′-5′ phosphodiester linkage on RNA structure and folding.

Main Methods:

• In vitro selection was employed to discover RNA-ligating deoxyribozymes.
• Ligation reactions were optimized for yield and kinetics under varying conditions (pH, temperature).
• Nondenaturing gel electrophoresis was used to analyze the folding of a ligated structured RNA domain.

Main Results:

• Mg(2+)-dependent deoxyribozymes were identified, achieving 50-60% yield of ligated RNA in overnight incubations.
• Faster ligation (40-50% yield in 1 hour) was observed at pH 9.0.
• A 2′-5′ linkage introduced into the Tetrahymena group I intron P4-P6 domain did not significantly disrupt its Mg(2+)-dependent folding.

Conclusions:

• Novel deoxyribozymes efficiently catalyze the formation of 2′-5′ RNA phosphodiester linkages.
• The introduced non-native 2′-5′ linkage is compatible with RNA secondary and tertiary structure formation.
• These deoxyribozymes are valuable tools for preparing site-specifically modified RNAs for structure-function investigations.