NMR Relaxation Footprinting: The [Cr(NH₃)₆]³⁺ Cation as a Probe for Drug Binding Sites on Oligonucleotides

Abstract

[Cr(NH₃)₆] (N0₃)₃ was used to probe structural features of the oligonucleotide, d(ATGCdCAT)₂ (numbering of strand: 5′ A₁T₂G₃C₄G₅C₆A₇T₈ 3′) and to footprint the binding site of actinomycin D(ActD) in the unique 2:1 ActD/d(ATGCGCAT)₂ complex by proton longitudinal relaxation (T₁) studies. Longitudinal relaxation rates (1/T₁) of the ¹H NMR signals were measured before and after the addition of the [Cr(NH₃)₆]³⁺ solution to determine the paramagnetic longitudinal relaxation rate (1/T_1p = 1/T_1(Cr) — l/T_1(noCr)). The chromium complex seems to prefer the center of the duplex, since signals for protons on nucleotides in the center of the duplex have the largest 1/T_1ps. Larger 1/T′_1p values are observed for signals of major groove base protons on G₃, C₄, G₅ and C₆ ancfalso for signals of deoxyribose H1′ and H3′ protons which are close to the phosphate backbone (H3′ closer than H1′). We believe that electrostatic forces and hydrogen bonding are the main interactions between the chromium hexaammine cation and d(ATGCGCAT)₂. To interpret our data we used distances from computer-generated models for five major binding modes. Four of these involved hydrogen bonding of the ammonia ligands to various sites on the oligonucleotide (phosphate oxygens, base oxygens and base nitrogens). One mode involved the approach of the [Cr(NH₃)₆]³⁺ cation into the minor groove of the duplex with no hydrogen bonding. Neither a single binding mode nor an equal weighting of all binding modes appeared to explain the results. Two modes appeared to have the greatest influence. One involved major groove interstrand binding at G₃ and G₅. The other involved interaction of the cation with a single phosphate group, with all phosphate groups exhibiting this binding mode. Other modes most likely do occur, but from modeling studies these modes appear to be less important.