Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment

Abstract

Double electron-electron resonance (DEER) is an electron paramagnetic resonance (EPR) technique used to determine distance distributions in the nanometer range between spin labels by measuring their dipole-dipole interactions. Here we describe how in-cell DEER can be applied to spin-labeled DNA sequences to unravel their conformations in living cells by long-range distance measurements in cellula. As EPR detects unpaired electron spins only, diamagnetic molecules provide no background and do not reduce detection sensitivity of the specific signal. Compared with in-cell NMR spectroscopy, low concentrations of spin-labeled molecules can be used owing to the higher sensitivity of EPR per spin. This protocol describes the synthesis of the spin labels, their introduction in DNA strands, the injection of labeled DNA solutions in cells and the performance of in-cell EPR measurements. Completion of the entire protocol takes ~20 d.