Alignment of Gold Clusters on DNA via a DNA-Recognizing Zinc Finger-Metallothionein Fusion Protein

作者：Ariyasu Shinya; Onoda Akira; Sakamoto Ryota; Yamamura Takeshi^*

来源：Bioconjugate Chemistry, 2009, 20(12): 2278-2285.

DOI：10.1021/bc9002713

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 alpha 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-MT alpha (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 (Cd(2 ) particularly forms a Cd(4)(Cys)(9) cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5'-GGGGGG-3' (GO bp sequence in the presence of Zn(2 ) based on the amino acid sequence encoded in the ZF domain. The titration of Au(11)(PPh(3))(9)Cl(3) (Au11) into the solution of 1 in the presence of Zn(2 ) revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au(11)-containing conjugate, ZF(Zn)ZF(Zn)-MT alpha(Au(11)) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au(11) core in the form of Au(11)(PPh(3))(4)(S-Cys)(6) without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MT alpha(Au(11)) recognizes the bp sequence G(6) 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 G(6) encoded in the dx DNA.

• 出版日期2009-12