MORIMURA ET AL. VOL. 7 ’ NO. 12 ’ 10733–10740 ’ 2013 www.acsnano.org 10733 November 06, 2013 C 2013 American Chemical Society Nano-Analysis of DNA Conformation Changes Induced by Transcription Factor Complex Binding Using Plasmonic Nanodimers Hiroyuki Morimura, † Shin-Ichi Tanaka, ‡ Hidekazu Ishitobi, †,‡ Tomoyuki Mikami, ‡ Yusuke Kamachi, ‡ Hisato Kondoh, ‡ and Yasushi Inouye †,‡,§, * † Department of Applied Physics, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, ‡ Graduate School of Frontier Biosciences, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan, and § Photonics Advanced Research Center, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan T he local surface plasmon is a resonant phenomenon of free electrons that collectively oscillate in noble metal nanoparticles (hereafter referred to as “NP”) and the electric field of incident light. 15 Since metal NPs scatter incident light strongly at the plasmon resonant wave- length, metallic NPs have been widely used as biomolecular tags in single-molecular tracking studies. 6 As the proximity of two metallic NPs shortens, the frequency of the collective oscillation decreases because of the electrostatic attraction due to the sur- face charge, and the plasmon resonant wavelength lengthens. 7,8 Therefore, the gap distance between two NPs can be determined at less the diffraction limit by measuring the plasmon resonant wave- lengths. Already, this strategy has been used to detect short DNA-associated events such as DNA double-strand hybridization 911 and the enzymatic cleavage of DNA. 12,13 In this paper, we applied the plasmon reso- nance for pairs of gold NPs (hereafter re- ferred to as “gold nanodimers”) to study a more elaborate process: DNA conformation changes induced by the binding of tran- scription factor complexes. In practice, nano- dimers are formed by bridging two gold NPs using double-stranded DNAs that have binding sequences for the transcription fac- tor complexes. Here, DNA conformation changes induced by the binding of a single transcription factor or transcription factor complex were measured as shifts in the plasmon resonant wavelength, which indi- cated shortening in the gap distance as the DNA bends. The transcription factor SOX2 (SRY- related-HMG-box 2) and other SOX proteins * Address correspondence to ya-inouye@ap.eng.osaka-u.ac.jp. Received for review July 16, 2013 and accepted November 6, 2013. Published online 10.1021/nn403625s ABSTRACT The plasmon resonant wavelength for a pair of gold nano- particles, or gold nanodimer, increases inversely with the gap distance between the two nanoparticles. Taking advantage of this property, we performed nanoscale measurements of DNA conformation changes induced by transcription factor binding. Gold nanoparticles were bridged by double- stranded DC5 DNA that included binding sequences for the transcription factors SOX2 and PAX6, which interact on the DC5 enhancer sequence and activate transcription. The gold nanodimers bound by SOX2 shifted the plasmon resonant wavelength from 586.8 to 604.1 nm, indicating that SOX2 binding induces DNA bending. When the SOX2 formed a ternary complex with PAX6 on DC5, the plasmon resonant wavelength showed a further shift to 611.6 nm, indicating additional bending in the DC5 sequence. Furthermore, we investigated DNA conformation changes induced by SOX2 and PAX6 on the DC5-con sequence, which is a consensus sequence of DC5 for the PAX6 binding region that strengthens the PAX6 binding but at the same time disrupts SOX2PAX6-dependent transcriptional activation. When the PAX6 binding sequence in DC5 was altered to DC5-con, the plasmon resonant wavelength shifted much less to 606.5 nm, which is more comparable to the 603.9 nm by SOX2 alone. These results demonstrate that SOX2PAX6 cobinding induces a large conformation change in DC5 DNA. KEYWORDS: local surface plasmon . nanodimer . DNA conformation change . transcription . SOX2 . PAX6 ARTICLE