Force spectroscopy of Rev-peptide–RRE interaction from HIV-1

Abstract

The specific interaction of the RNA recognition motif of Rev and its viral mRNA target, RRE, has been demonstrated for the first time at the single-molecule level by atomic-force-microscope based single-molecule-force-spectroscopy (AFM-SMFS). The approach reveals details of the dissociation pathway and contribution of base mutations. Specific RNA-protein interaction is efficiently blocked by the RNA binding agent neomycin, showing the potential of AFM-SMFS as an efficient tool for singlemolecule drug screening of RNA targets. Furthermore, we show the importance of surface chemistry in AFM-SMFS of RNA-protein interaction, in particular the influence of polymer linkers.

Quantitative Evaluation of Viral Protein Binding to Phosphoinositide Receptors and Pharmacological Inhibition

“…The calculated K a value of 4.88 × 10 5 M –1 agrees well with literature values obtained by conventional biochemical methods (1–7 × 10 5 M –1 ). 62 , 63 Of note, Živković et al 64 previously attempted to use AFM force spectroscopy to measure the effect of 100 mM neomycin inhibitor on peptide–RNA binding interactions, but they still detected some specific peptide–RNA binding events despite the neomycin concentrations being well in excess (by a factor of 5 × 10 4 ) of the corresponding dissociation constant. From this viewpoint, our findings reveal for the first time that, by utilizing the AH–PI(4,5)P 2 binding interaction as a competitive probe, AFM force spectroscopic measurements can distinguish the extent of neomycin’s inhibitory activity at drug concentrations 5 orders of magnitude lower than previously observed in other AFM force spectroscopic systems and provide quantitative readouts of equilibrium binding constants for phosphoinositide–small-molecule interactions.…”

Atomic force microscopy of RNA: State of the art and recent advancements

“…Heus et al investigated the Rev-peptide-RRE interaction for the first time by AFM based force spectroscopy [51]. For this purpose the Rev peptide was immobilized to the AFM tip and the RRE RNA to the surface.…”

Imaging and force probing RNA by atomic force microscopy

“…Heus and co-workers elucidated the Rev-peptide-RRE interaction for the first time by AFM based force spectroscopy [53] which was investigated by AFM imaging first as described above. By AFM force spectroscopy the authors elucidated the RNA-protein interaction after tethering the peptide to the AFM tip and the RRE RNA to the surface.…”