Multiple-quantum HCN-CCH-TOCSY experiment for 13C/15N labeled RNA oligonucleotides

Area of Science:

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

Background:

• Assigning RNA ribose resonances is crucial for understanding RNA structure and function.
• Existing COSY- and TOCSY-type experiments rely on 13C or 1H for spin system differentiation.
• A need exists for alternative, potentially more sensitive, methods for RNA resonance assignment.

Purpose of the Study:

• To introduce and validate a novel multiple-quantum 3D HCN-CCH-TOCSY experiment.
• To demonstrate the utility of this experiment for assigning RNA ribose resonances.
• To compare its performance against existing NMR techniques.

Main Methods:

• Development and implementation of a 3D HCN-CCH-TOCSY NMR experiment.
• Utilizing the chemical shift dispersion of N1 (pyrimidine) and N9 (purine) for ribose spin system identification.
• Application of the experiment to a 23-mer RNA aptamer complexed with neomycin.

Main Results:

• The multiple-quantum 3D HCN-CCH-TOCSY experiment successfully assigns RNA ribose resonances.
• The method effectively distinguishes ribose spin systems by leveraging specific nitrogen chemical shifts.
• Sensitivity was enhanced by an average factor of 2 compared to the single-quantum version in the tested complex.

Conclusions:

• The presented multiple-quantum 3D HCN-CCH-TOCSY experiment is a valuable alternative for RNA ribose resonance assignment.
• This technique offers improved sensitivity and a distinct approach to spin system differentiation.
• It holds promise for advancing RNA structural studies, particularly for complexed systems.