Assignment methodology for larger RNA oligonucleotides: application to an ATP-binding RNA aptamer

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Uniform 13C, 15N labeling simplifies NMR assignments for small RNA oligonucleotides, adapting protein study methods for ribose spin systems.
• Sequential assignment of RNA protons, both exchangeable and nonexchangeable, requires specialized NMR experiments.
• Challenges in assigning larger RNAs include short carbon relaxation times and extensive spectral overlap.

Purpose of the Study:

• To overcome limitations in NMR assignment of larger RNAs.
• To develop and apply a strategy combining base-selective labeling and RNA variants for enhanced structural assignment.
• To achieve complete sequential assignments for a complex RNA-ligand system.

Main Methods:

• Utilized base-type selectively 13C, 15N-labeled RNA.
• Employed related RNAs with base substitutions.
• Applied these methods to a 36-nucleotide ATP-binding RNA aptamer in complex with AMP.

Main Results:

• Successfully obtained complete sequential proton (1H) assignments for the RNA aptamer-AMP complex.
• Achieved the majority of carbon (13C) and nitrogen (15N) assignments.
• Demonstrated the efficacy of the combined labeling and variant RNA strategy for complex RNA structures.

Conclusions:

• The combined approach of base-selective labeling and RNA variants effectively addresses spectral overlap and relaxation time issues in NMR.
• This strategy significantly enhances the ability to perform unambiguous sequential assignments in larger RNA molecules.
• The method provides a powerful tool for detailed structural characterization of RNA-ligand complexes.