Dynamic Nuclear Polarization with Electron Decoupling in Intact Human Cells and Cell Lysates

Area of Science:

• Nuclear Magnetic Resonance Spectroscopy
• Dynamic Nuclear Polarization (DNP)
• Biophysical Chemistry

Background:

• Dynamic nuclear polarization (DNP) significantly enhances Nuclear Magnetic Resonance (NMR) sensitivity by transferring electron spin polarization to nuclei.
• Undesired interactions between DNP polarizing agents (radicals) and nearby nuclear spins can reduce signal quality in biological samples.
• Electron decoupling (eDEC) and chirped microwave pulses mitigate these detrimental effects in model systems but haven't been applied to intact cells.

Purpose of the Study:

• To investigate the application of electron decoupling (eDEC) in intact human cells and cell lysates for enhanced DNP-NMR sensitivity.
• To evaluate the efficiency of eDEC across various temperatures, solvents, and with different trityl radical derivatives.
• To demonstrate exceptionally fast ¹H T_1DNP relaxation times in intact human cells.

Main Methods:

• Synthesis and application of a novel methylated trityl radical for DNP in intact human cells.
• Implementation of electron decoupling (eDEC) techniques with frequency-chirped microwave pulses.
• Measurement of ¹H T_1DNP relaxation times and ¹³C signal intensity enhancements at varying temperatures (90 K and 6 K).

Main Results:

• Achieved exceptionally fast ¹H T_1DNP times of 200 ms at 90 K and 300 ms at 6 K in intact human cells.
• Demonstrated successful application of eDEC in intact human cells and both human and bacterial cell lysates.
• Observed significant ¹³C signal intensity increases with eDEC: 8% (90 K, intact cells), 10% (90 K, lysates), 15% (6 K, intact cells), and 39% (6 K, lysates).

Conclusions:

• Electron decoupling (eDEC) is effective in enhancing DNP-NMR sensitivity in intact human cells and cell lysates.
• The combination of eDEC, chirped microwave pulses, and low temperatures (approaching 6 K) is a powerful strategy for rapid, high-sensitivity DNP in complex biological environments.
• This approach holds significant promise for advancing NMR applications in cellular and biomolecular studies.