title: Alignment of gold clusters on DNA via a DNA-recognizing zinc finger-metallothionein fusion protein.
abstract: The complementary recognition of base pairs (bp) is the major strategy in the "DNA lithography" of gold (Au)clusters or nanoparticles, where single-stranded DNAs sulfurized at their termini are generally used to bind Au clusters or nanoparticles. In this report, we discuss a new material that can be used to locate Au clusters on the desired positions of DNA. For this purpose, we combined a two-domain zinc finger (ZF) and the analogue of R domain of rat's liver metallothionein (MT) to utilize the DNA-recognizing ability of ZF motifs and the heavy metal binding ability of MTs, and prepared an artificial fusion protein, ZFZF-MTalpha (1). Titration experiments monitored by absorption and circular dichroism spectroscopies, as well as an electrophoretic mobility shift assay and quantification using 5,5'-dithiobis(2-nitrobenzoic acid), clarified that (1) the ZF domain traps two divalent metal ions to fold in a ZF structure with M(Cys)2(His)2 (M = Co, Zn, and Cd) coordination units, (2) the MT domain traps metal ions to form clusters (Cd2+ particularly forms a Cd4(Cys)9 cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5'-GGGGGG-3' (G6) bp sequence in the presence of Zn2+ based on the amino acid sequence encoded in the ZF domain. The titration of Au11(PPh3)8Cl3 (Au11) into the solution of 1 in the presence of Zn2+ revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au11-containing conjugate, ZF(Zn)ZF(Zn)-MTalpha(Au11) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au11 core in the form of Au11(PPh3)4(S-Cys)6 without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MTR(Au11) recognizes the bp sequence G6 with K(d1) = 450 nM, while simultaneously forming a dimer on the DNA with K(d2) = 200 nM. TEM experiments showed that the conjugates form parallelograms or triangles (defective parallelograms) on a double crossover (dx) DNA, according to the positions of G6 encoded in the dx DNA.
authors: Department of Chemistry, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan.
authors: Shinya
authors: S
authors: Ariyasu
authors: Akira
authors: A
authors: Onoda
authors: Ryota
authors: R
authors: Sakamoto
authors: Takeshi
authors: T
authors: Yamamura
mesh_terms: D000465:Algorithms; D000818:Animals; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006046:Gold / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry*; D008668:Metallothionein / Q000737:chemistry*; D010316:Particle Size; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D013499:Surface Properties; D016335:Zinc Fingers
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D006046:Gold; D004247:DNA; D008668:Metallothionein