High resolution footprinting ofEcoRI and distamycin with Rh(phi)₂(bpy)³⁺, a new photofootprinting reagent

Abstract

The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)2(bpy)3+, cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootpring reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5-7 base pair footprint for the drug at its A6 binding site and a 10-12 base pair footprint for the enzyme centered at its recognition site (5'-GAATTC-3'). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg2+ or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.

Role of Forward Translocation in Nucleoside Triphosphate Phosphohydrolase I (NPH I)-mediated Transcription Termination of Vaccinia Virus Early Genes

“…The results of these studies indicated that this distance is 17 bp (see supplemental material). Moreover, prior studies have determined the distance between the leading edge of vRNAP and the active site to be 15 bp (39), and the distance between the upstream edge of EcoRI* and the first G in GAATTC to be 2 bp (39). The sum of these two distances agrees well with our estimate of 17 bp.…”

Perfect Strangers: Inorganic Photochemistry and Nucleic Acids

“…An advantage of the diplatinum complex is that it does not produce a diffusible intermediate such as ؒOH, which is easily quenched by reagents commonly used to maintain the native form of the protein, such as DTT or glycerol. The complex Rh(phi) 2 (bpy) 3+ also exhibits a sequence-neutral photocleavage pattern and has been used to footprint the binding of both EcoRI and distamycin on a restriction fragment (44).…”

Fast photoinduced electron transfer through DNA intercalation.

“…28; J. G. Collins, T. P. Shields, and J.K.B., unpublished data). Phi complexes of rhodium cleave DNA and RNA upon photoactivation and thus are also useful as probes of higher-order structures in nucleic acids and as high-resolution DNA photofootprinting reagents (29)(30)(31)(32)(33)(34)(35). The absorption spectrum ofRu(phen)2dppz2+ is characterized by a metal-to-ligand charge transfer transition centered on the dppz ligand, and the emission from this state in nonaqueous solution can be quenched by electron acceptors (36).…”