Cu(II)-Based Paramagnetic Probe to Study RNA–Protein Interactions by NMR

Paramagnetic NMR techniques allow
for studying three-dimensional structures of RNA–protein complexes.
In particular, paramagnetic relaxation enhancement (PRE) data can
provide valuable information about long-range distances between different
structural components. For PRE NMR experiments, oligonucleotides are
typically spin-labeled using nitroxide reagents. The current work
describes an alternative approach involving a Cu­(II) cyclen-based
probe that can be covalently attached to an RNA strand in the vicinity
of the protein’s binding site using “click” chemistry.
The approach has been applied to study binding of HIV-1 nucleocapsid
protein 7 (NCp7) to a model RNA pentanucleotide, 5′-ACGCU-3′.
Coordination of the paramagnetic metal to glutamic acid residue of
NCp7 reduced flexibility of the probe, thus simplifying interpretation
of the PRE data. NMR experiments showed attenuation of signal intensities
from protein residues localized in proximity to the paramagnetic probe
as the result of RNA–protein interactions. The extent of the
attenuation was related to the probe’s proximity allowing us
to construct the protein’s contact surface map.