An Unexpected Adenosine-Alkylating Ribozyme Emerged by Target Site Relocation during in Vitro Selection

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Therapeutics

Background:

• RNA-alkylating ribozymes are essential tools for post-transcriptional RNA modification.
• They facilitate site-specific installation of various chemical groups for RNA analysis.
• Existing ribozymes have limitations in target specificity and modification capabilities.

Purpose of the Study:

• To discover and characterize novel RNA-alkylating ribozymes with improved properties.
• To investigate the structure-activity relationship of new ribozyme variants.
• To expand the toolbox for site-specific RNA modification.

Main Methods:

• In vitro selection combined with high-throughput sequencing (RZ-seq).
• Biochemical analyses including in-line probing and mutational profiling.
• Characterization of ribozyme variants for catalytic activity and target specificity.

Main Results:

• Identification of a highly active RNA-alkylating catalytic RNA (RACR) targeting adenosine.
• RACR utilizes modified guanine cofactors to alkylate the N1 position of adenosine within the NAYN motif.
• RACR demonstrates a distinct catalytic core and RNA target engagement mechanism compared to MTR1.
• The ribozyme can be directed to modify diverse RNA molecules like tRNA and snRNA.

Conclusions:

• RACR represents a novel and versatile tool for post-synthetic RNA modification, expanding the ribozyme toolbox.
• The study provides insights into in vitro selection dynamics for future ribozyme evolution.
• The RZ-seq method facilitates efficient discovery and characterization of novel catalytic RNAs.