RNA circularization preserves dynamics and enables in-cell relaxation NMR.

Area of Science:

• Structural Biology
• Biochemistry
• Molecular Biophysics

Background:

• RNA dynamics are crucial for function but difficult to study in cells due to RNase degradation.
• Nuclear Magnetic Resonance (NMR) spectroscopy offers atomic-level insights into RNA dynamics but is limited to in vitro studies.
• Current methods to stabilize RNA for in-cell studies often require chemical modifications.

Purpose of the Study:

• To develop a method for stabilizing unmodified RNA to enable in-cell NMR studies.
• To investigate the impact of RNA circularization on RNA stability, structure, and dynamics.
• To explore RNA dynamics and potential allosteric mechanisms within a cellular environment.

Main Methods:

• Development of an enzymatic pipeline for circularizing RNA hairpins.
• Assessment of circular RNA stability in cellular lysate using NMR.
• Characterization of secondary structure and excited-state dynamics of circularized RNAs via NMR spectroscopy.
• Measurement of imino proton relaxation rates for in-cell NMR studies.

Main Results:

• Circularized RNA exhibited enhanced stability (>24 h in lysate) without chemical modifications.
• Circularization preserved secondary structure and native dynamics for diverse RNA constructs.
• Observed transfer of dynamics across RNA helices in circularized RNAs, suggesting allosteric cooperativity.
• Achieved reproducible in-cell NMR measurements of relaxation rates on unmodified circular RNA.

Conclusions:

• RNA circularization is a versatile strategy for enabling quantitative in-cell NMR studies of RNA structure and dynamics.
• This method overcomes limitations of RNA degradation in cellular environments.
• The findings suggest a potential mechanism for long-range cooperativity in RNA function.