Poster abstracts

Poster number 38 submitted by Jeff Joyner

Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates

Jeff C. Joyner (Ohio State Biochemistry Program), James A. Cowan (Chemistry Department)

Abstract:
A series of compounds that target reactive metal-chelates to the HIV-1 Rev Response Element (RRE) mRNA have been synthesized, and both dissociation constants and chemical reactivity toward HIV RRE RNA determined. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine sidechain of a Rev-derived peptide with either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(II), Co(II), Ni(II), and Cu(II) such that the arginine-rich Rev peptide could mediate localization of the metal-chelates to the Rev peptide’s high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high affinity RRE-binding by all 22 metal-chelate-Rev species, with dissociation constants ranging from ~ 0.2 – 16 nM, indicating little to no loss of RRE affinity due to the coupling of the metal-chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RRE modification by each metal-chelate-Rev species were determined and varied from ~ 0.28 – 4.9 nM/min, but were optimal for Cu(II)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs. NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between that of the two principal coreagents for metal-promoted formation of reactive oxygen species: E° for ascorbate/ascorbyl radical = -66 mV; E° for hydrogen peroxide/hydroxyl radical = 380 mV. This class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 Rev response element mRNA.

Keywords: Metallotherapeutics, HIV, Artificial Nuclease