Sequence-Selective DNA Cleavage by a Chimeric Metallopeptide

Abstract

A chimeric metallopeptide derived from the sequences of two structurally superimposable motifs was designed as an artificial nuclease. Both DNA recognition and nuclease activity have been incorporated into a small peptide sequence. P3W, a 33-mer peptide comprising helices alpha2 and alpha3 from the engrailed homeodomain and the consensus EF-hand Ca-binding loop binds one equivalent of lanthanides or calcium and folds upon metal binding. The conditional formation constants (in the presence of 50 mM Tris) of P3W for Eu(III) (K(a) = (2.1 +/- 0.1) x 10(5) M(-1)) and Ce(IV) (K(a) = (2.6 +/- 0.1) x 10(5) M(-1)) are typical of isolated EF-hand peptides. Circular dichroism studies show that 1:1 CeP3W is 26% alpha-helical and EuP3W is up to 40% alpha-helical in the presence of excess metal. The predicted helicity of the folded peptide based on helix length and end effects is about 50%, showing the metallopeptides are significantly folded. EuP3W has considerably more secondary structure than our previously reported chimeras (Welch, J. T.; Sirish, M.; Lindstrom, K. M.; Franklin, S. J. Inorg. Chem. 2001, 40, 1982-1984). Eu(III)P3W and Ce(IV)P3W nick supercoiled DNA at pH 6.9, although EuP3W is more active at pH 8. CeP3W cleaves linearized, duplex DNA as well as supercoiled plasmid. The cleavage of a 5'-(32)P-labeled 121-mer DNA fragment was followed by polyacrylamide gel electrophoresis. The cleavage products are 3'-OPO(3) termini exclusively, suggesting a regioselective or multistep mechanism. In contrast, uncomplexed Ce(IV) and Eu(III) ions produce both 3'-OPO(3) and 3'-OH, and no evidence of 4'-oxidative cleavage termini with either metal. The complementary 3'-(32)P-labeled oligonucleotide experiment also showed both 5'-OPO(3) and 5'-OH termini were produced by the free ions, whereas CeP3W produces only 5'-OPO(3) termini. In addition to apparent regioselectivity, the metallopeptides cut DNA with modest sequence discrimination, which suggests that the HTH motif binds DNA as a folded domain and thus cleaves selected sequences. The de novo artificial nuclease LnP3W represents the first small, underivatized peptide that is both active as a nuclease and sequence selective.

Lanthanide(III) Complexes with Two Hexapeptides Incorporating Unnatural Chelating Amino Acids: Secondary Structure and Stability

“…[8] Imperiali and co-workers optimized the sequence of lanthanide-binding tags (LBTs) by using screening methods starting from Ca 2 + -binding sites to obtain the highest possible affinity. [9,10] Abstract: Unnatural metal-chelating amino acids bearing aminodiacetate side-chains have been introduced into two hexapeptides to obtain efficient lanthanide-binding peptides.…”

Studies on DNA cleavage and antimicrobial screening of transition metal complexes of 4-aminoantipyrine derivatives of N2O2 type

“…Among the different therapeutic strategies to eradicate cancer cells through DNA damage, the view of using small water soluble transition metal complexes, capable of oxidative or hydrolytic DNA cleavage as anticancer drugs is a challenging issue in bioinorganic chemistry [24,25]. Many transition metal complexes with vanadium [26], iron [27], copper [28,29], cobalt [30], lanthanides [31,32] and also actinides [33] have been reported as efficient DNA cleavage agents with or without sequence specificity, moreover the ligand or the metal in these complexes can be varied in an easily controlled manner to facilitate the individual applications [34].…”

Unique Ligand‐Based Oxidative DNA Cleavage by Zinc(II) Complexes of Hpyramol and Hpyrimol

“…[4,5] Among the different therapeutic strategies to eradicate cancer cells through DNA damage, the view of using small water-soluble transition-metal complexes, capable of oxidative or hydrolytic DNA cleavage as anti-cancer drugs, is a challenging topic in bioinorganic chemistry. [6,7] Many transition-metal complexes with V, [8] Fe, [9] Cu, [10,11] Co, [12] lanthanides, [13,14] and also actinides [15] have been reported as efficient DNA cleaving agents with or without sequence specificity. Of these metals, zinc is the second most abundant transition-metal ion present in the human body, [16,17] and is in the active sites of several enzymes such as superoxide dismutase, proteins containing zinc finger motifs and zinc hydrolases, in particular, owing to its Lewis acid character.…”