In vitro selection of self-cleaving RNAs with a low pH optimum

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Catalysis

Background:

• Ribonucleic acid (RNA) plays crucial roles in gene expression and catalysis.
• The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process is a powerful method for discovering functional nucleic acids.
• Understanding RNA's chemical reactivity is key to deciphering its biological functions and origins.

Purpose of the Study:

• To select and characterize RNA molecules capable of rapid, site-specific cleavage at low pH.
• To investigate the reaction mechanism, including cofactor requirements and kinetics.
• To explore the implications of such autocatalytic RNA cleavage for prebiotic chemistry.

Main Methods:

• In vitro selection (SELEX) to isolate RNA molecules with specific cleavage properties.
• Biochemical assays to determine reaction kinetics and optimal conditions (pH, ions).
• Analysis of cleavage products to identify the resulting chemical structures.

Main Results:

• Successfully selected RNA molecules that undergo rapid, site-specific cleavage at acidic pH.
• Cleavage is an intramolecular reaction, independent of RNA concentration.
• The reaction is inhibited by divalent metal ions, spermine, and high monovalent ion concentrations, requiring no metal cofactors.
• Cleavage produces a 2',3'-cyclic phosphate at the 3' end and a 5'-hydroxyl group.
• Optimal cleavage occurs around pH 4.0, with a rate constant of 1.1 min-1 at pH 5.0 and room temperature.

Conclusions:

• Identified novel autocatalytic RNA molecules with unique low-pH cleavage activity.
• The metal-ion-independent nature of this cleavage expands the known repertoire of RNA catalysis.
• These findings suggest plausible mechanisms for RNA-based chemistry on the prebiotic Earth.