Optimising in-cell NMR acquisition for nucleic acids.

Area of Science:

• Structural Biology
• Biophysics
• Cellular NMR Spectroscopy

Background:

• In-cell NMR spectroscopy is vital for understanding nucleic acid structure and function in native cellular environments.
• Challenges in human cell lines, including sample degradation and low signal intensity, limit current in-cell NMR applications.
• Broad line widths in cellular environments reduce the obtainable structural and dynamic information from NMR experiments.

Purpose of the Study:

• To optimize the detection of imino proton signals for double-stranded DNA (dsDNA) in HeLa cells using selective excitation.
• To enable reproducible quantification of in-cell selective longitudinal relaxation times (selT1) for nucleic acids.
• To enhance signal gain per unit time and shorten measurement duration for in-cell NMR experiments.

Main Methods:

• Utilized selective excitation techniques for imino proton signal detection in dsDNA within HeLa cells.
• Developed and optimized proton pulse sequences for intracellular selT1 measurements.
• Implemented experimental controls including intracellular quantification and supernatant measurements for data validation.

Main Results:

• Successfully optimized selective excitation for enhanced detection of DNA imino proton signals in HeLa cells.
• Demonstrated reproducible quantification of intracellular selT1, showing reduced values compared to in vitro conditions.
• Achieved shortened measurement times and enhanced signal gain per unit time compared to conventional methods.

Conclusions:

• Optimized selective excitation offers advantages over traditional methods like jump-return water suppression for in-cell NMR.
• Reduced intracellular selT1 values indicate interactions within the complex cellular environment affecting DNA dynamics.
• Robust and rapid in-cell NMR experiments are crucial for advancing the study of nucleic acid structure, dynamics, and function.