Targeted Cleavage of HIV RRE RNA by Rev-Coupled Transition Metal Chelates
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- 作者：Jeff C. Joyner ; J. A. Cowan
- 刊名：Journal of the American Chemical Society
- 出版年：2011
- 出版时间：June 29, 2011
- 年：2011
- 卷：133
- 期：25
- 页码：9912-9922
- 全文大小：1051K
- 年卷期：v.133,no.25(June 29, 2011)
- ISSN：1520-5126
文摘
A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. 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 side chain of a Rev-derived peptide by 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 Fe2+, Co2+, Ni2+, and Cu2+ such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide鈥檚 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 RNA binding by all 22 metal-chelate-Rev species, with KD values ranging from 0.2 to 16 nM, indicating little to no loss of RNA 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 RNA modification by each metal-chelate-Rev species were determined and varied from 0.28 to 4.9 nM/min but were optimal for Cu2+-NTA-Rev. Metal-chelate reduction potentials were determined and varied from 鈭?28 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 those of the two principal co-reagents for metal-promoted formation of reactive oxygen species: E掳ascorbate/ascorbyl鈥塺adical = 鈭?6 mV and E掳H2O2/hydroxyl鈥塺adical = 380 mV. Given the variety of oxidative activities of these metal complexes and their high-affinity binding to the targeted RRE mRNA following coupling to the Rev peptide, this class of metal-chelate-Rev derivatives constitutes a promising step toward development of multiple-turnover reagents for selective eradication of HIV-1 RRE mRNA.