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Peptide nucleic acids (PNAs) are a promising group of synthetic analogues of DNA and RNA that offer several distinct advantages over the naturally occurring nucleic acids for applications in biosensing, drug delivery, and nanoelectronics. Because of its structural differences from DNA/RNA, methods to analyze and assess the structure, conformations, and dynamics are needed. In this work, we develop synergistic techniques for the study of the PNA conformation. We use CuQ2, a Cu(II) complex with 8-hydroxyquinoline (HQ), as an alternative base pair and as a spin label in electron paramagnetic resonance (EPR) distance methods. We use molecular dynamics (MD) simulations with newly developed force field parameters for the spin labels to interpret the distance constraints determined by EPR. We complement these methods by UV–vis and circular dichroism measurements and assess the efficacy of the Cu(II) label on a PNA duplex whose backbone is based on aminoethylglycine and a duplex with a hydroxymethyl backbone modification. We show that the Cu(II) label functions efficiently within the standard PNA and the hydroxymethyl-modified PNA and that the MD parameters may be used to accurately reproduce our EPR findings. Through the combination of EPR and MD, we gain new insights into the PNA structure and conformations as well as into the mechanism of orientational selectivity in Cu(II) EPR at X-band. These results present for the first time a rigid Cu(II) spin label used for EPR distance measurements in PNA and the accompanying MD force fields for the spin label. Our studies also reveal that the spin labels have a low impact on the structure of the PNA duplexes. The combined MD and EPR approach represents an important new tool for the characterization of the PNA duplex structure and provides valuable information to aid in the rational application of PNA at large.