In vitro evolution of ribonucleases from expanded genetic alphabets

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Nucleic acids (NAs) are crucial for information storage and catalysis, but their catalytic power is limited compared to proteins.
• Standard NAs use four nucleotide building blocks, restricting their catalytic repertoire.
• Expanding the NA alphabet offers potential for enhanced catalytic functions.

Purpose of the Study:

• To assess the evolutionary potential of nucleic acid libraries with six nucleotide building blocks for catalysis.
• To compare the RNA-cleavage activity of libraries with standard versus expanded nucleotide sets.

Main Methods:

• In vitro selection experiments were conducted on two nucleic acid libraries: one with four standard nucleotides and another with six, including two from an artificially expanded genetic information system (AEGIS).
• The catalytic activity, specifically RNA-cleavage, was compared between the two libraries.

Main Results:

• DNA libraries with increased chemical diversity (six nucleotides) demonstrated catalytic activity for RNA cleavage one order of magnitude higher than standard four-nucleotide libraries.
• Evolved AEGISzymes utilize a general acid-base catalytic mechanism, similar to ribonuclease A and modified DNAzymes.
• The expanded alphabet provides a richer reservoir for discovering novel catalysts.

Conclusions:

• Nucleic acids built from expanded genetic alphabets, like AEGIS, significantly enhance catalytic capabilities.
• AEGISzymes represent a new class of catalysts with potential biomedical applications.
• Increased chemical diversity in nucleic acid libraries expands their potential as catalytic reservoirs.