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PELDOR Measurements on Nitroxide-Labeled Oligonucleotides.

Tobias Hett¹, Olav Schiemann²

¹Institute of Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University, Bonn, Germany.

Methods in Molecular Biology (Clifton, N.J.)

|February 28, 2022

Summary

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) offers insights into biomolecule structure. This study details setting up pulsed electron-electron double resonance (PELDOR) experiments for analyzing labeled oligonucleotides.

Keywords:

Double electron–electron resonance (DEER)Electron paramagnetic resonance (EPR)Electron spin resonance (ESR)Pulsed dipolar spectroscopy (PDS)Pulsed electron–electron double resonance (PELDOR)Spin label

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Published on: November 8, 2019

Area of Science:

Biophysical Chemistry
Biomolecular Structure Determination
Spectroscopy

Background:

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) is a key technique for studying biomolecular structure, dynamics, and function.
PDS methods yield distance distributions between spin centers, which can be intrinsic or introduced via site-directed spin labeling.
Pulsed electron-electron double resonance (PELDOR), also known as distance-based electron-echo (DEER), is the most common PDS experiment for measuring nanometer-scale interspin distances.

Purpose of the Study:

To provide a comprehensive, step-by-step protocol for conducting PELDOR experiments on commercially available pulsed EPR spectrometers.
To outline the essential data analysis procedures following PELDOR measurements.
To highlight common challenges and potential pitfalls encountered during PELDOR experiments.

Main Methods:

Detailed workflow for setting up PELDOR experiments on a pulsed EPR spectrometer.
Methodology for analyzing PELDOR data to extract structural information.
Application of site-directed spin labeling using nitroxide labels on oligonucleotides.

Main Results:

A practical guide for implementing PELDOR experiments is presented.
The study facilitates the structural investigation of oligonucleotides, including RNA-cleaving DNAzymes and their RNA targets.
The protocol enables the study of modified DNAzymes with diverse functions and targets by incorporating nitroxide-labeled nucleotides.

Conclusions:

The presented protocol simplifies the setup and execution of PELDOR experiments for biomolecular structure determination.
PELDOR is demonstrated as a valuable tool for studying the structural intricacies of oligonucleotides and their interactions.
This work supports the application of PELDOR in investigating the structure-function relationships of modified DNAzymes.