Displacement of Mn2+ from RNA by K+, Mg2+, neomycin B, and an arginine-rich peptide: indirect detection of nucleic acid/ligand interactions using phosphorus relaxation enhancement.
We have developed a novel method to study the interactions of nucleic acids with cationic species. The method, called phosphorus relaxation enhancement (PhoRE), uses (1)H-detected (31)P NMR of exogenous probe ions to monitor changes in the equilibrium between free Mn(2+) and Mn(2+) bound to the RNA. To demonstrate the technique, we describe the interactions of four RNA molecules with metal ions (K(+) and Mg(2+)), a small molecule drug (neomycin b), and a cationic peptide (RSG1.2). In each case, cationic ligand binding caused Mn(2+) to be displaced from the RNA. Free Mn(2+) was determined from its effect on the T(2) NMR relaxation rate of either phosphite (HPO(3)(2-)) or methyl phosphite (MeOPH, CH(3)OP(H)O(2-)). Using this method, the effects of [RNA] as low as 1 microM could be measured in 20 min of accumulation using a low field (200 MHz) instrument without pulsed field gradients. Cation association behavior was sequence and [RNA] dependent. At low [K(+)], Mn(2+) association with each of the RNAs decreased with increasing [K(+)] until approximately 40 mM, where saturation was reached. While saturating K(+) displaced all the bound Mn(2+) from a 31-nucleotide poly-uridine (U(31)), Mn(2+) remained bound to each of three hairpin-forming sequences (A-site, RRE1, and RRE2), even at 150 mM K(+). Bound Mn(2+) was displaced from each of the hairpins by Mg(2+), allowing determination of Mg(2+) dissociation constants (K(d,Mg)) ranging from 50 to 500 microM, depending on the RNA sequence and [K(+)]. Both neomycin b and RSG1.2 displaced Mn(2+) upon binding the hairpins. At [RNA] approximately 3 microM, RRE1 bound a single equivalent of RSG1.2, whereas neither RRE2 nor A-site bound the peptide. These behaviors were confirmed by fluorescence polarization using TAMRA-labeled peptide. At 2.7 microM RNA, the A-site hairpin bound a single neomycin b molecule. The selectivity of RSG1.2 binding was greatly diminished at higher [RNA]. Similarly, each hairpin bound multiple equivalents of neomycin at the higher [RNA]. These results demonstrate the utility of the PhoRE method for characterizing metal binding behaviors of nucleic acids and for studying RNA/ligand interactions.
Summers
Jack S
JS
Message Pharmaceuticals, Inc., 30 Spring Mill Road, Malvern, PA 19355, USA. jack@hhmi.umbc.edu
Shimko
John
J
Freedman
Fredric L
FL
Badger
Christopher T
CT
Sturgess
Michael
M
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry*; D001483:Base Sequence; D002412:Cations; D005454:Fluorescence Polarization; D005601:Framycetin / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D012313:RNA / Q000737:chemistry*
D002412:Cations; D008670:Metals; D010455:Peptides; D008345:Manganese; D005601:Framycetin; D012313:RNA; D001120:Arginine; D008274:Magnesium; D011188:Potassium