Efficient Assembly of Functional RNA by in Situ Phosphate Activation and Loop-Closing Ligation

Area of Science:

• Origin of Life Studies
• RNA Biochemistry
• Prebiotic Chemistry

Background:

• The emergence of ribozymes before efficient RNA replication is a key question in origin of life research.
• Nonenzymatic RNA ligation is a potential pathway, but requires high metal ion concentrations incompatible with early protocells.
• Oligoribonucleotide 5'-phosphate activation and competing hydrolysis limit ligation efficiency in aqueous environments.

Purpose of the Study:

• To investigate template-independent RNA assembly via loop-closing ligation using a prebiotic in situ phosphate activation approach.
• To overcome limitations of metal ion dependence and hydrolysis in nonenzymatic RNA ligation.
• To provide a viable strategy for synthesizing functional RNA molecules.

Main Methods:

• Investigated N-methylimidazole-intercepted phospho-Passerini reaction for 5'-phosphate activation.
• Utilized loop-closing ligation for hairpin RNA formation.
• Performed successive in situ activation/ligation steps to assemble a functional RNA Flexizyme.

Main Results:

• Demonstrated efficient hairpin RNA formation via loop-closing ligation using in situ phosphate activation.
• Showed the reaction proceeds without divalent metal ions and is effective at low millimolar concentrations of activating reagent.
• Identified a novel pathway involving N-imidoyl-N'-methylimidazolium intermediate leading to phosphorimidazolium species.
• Achieved near-quantitative yield in assembling a functional RNA Flexizyme through two successive ligation steps.

Conclusions:

• The developed method offers a viable pathway for the prebiotic origin of functional RNAs.
• This strategy provides a new approach for the chemical synthesis of long RNA molecules.
• The findings address the challenge of RNA ligation in early Earth conditions.