Nanobiotechnology DOI: 10.1002/anie.200602049 Reversible Binding of Fluorescent Proteins at DNA–Gold Nanoparticles** Pompi Hazarika, Florian Kukolka, and Christof M. Niemeyer* Protein-functionalized gold nanoparticles (AuNPs) are cur- rently attracting increasing interest as a result of their applications in the detection of antigens, which are specific biomarkers in disease diagnostics. [1,2] Protein–AuNPs have long been known as versatile probes in immunohistochemis- try and related techniques, [3] while mixed hybrid particles containing both nucleic acid fragments and proteins have recently been established, [4–7] because they offer additional functionality on account of the presence of both classes of biomolecular recognition elements. For instance, DNA– AuNPs containing a low number of antibody molecules have been successfully employed in the highly sensitive detection of a soluble pathogenic biomarker for Alzheimer)s disease. [6] The synthesis of DNA–protein hybrid particles has been achieved by either chemisorption of the sulfhydryl groups of proteins to AuNPs followed by saturation of the particles with thiolated DNA oligonucleotides, [5,6] or specific DNA hybrid- ization using DNA–AuNPs and appropriate protein conju- gates bearing the complementary DNA oligomer moiety. [4,7] Herein, we report the extension of the latter approach to render the binding of DNA–protein conjugates to DNA– AuNPs reversible. To this end, we employed the concept of DNA oligonucleotide strand displacement, which has fre- quently been used in the development of nanomechanical DNA devices, [8–14] and we have recently used this approach to reversibly switch the aggregation of DNA–AuNPs. [15] The realization of the DNA-directed immobilization and detach- ment of proteins to gold nanoparticles, which is depicted schematically in Figure 3a, would be advantageous over conventional chemisorption, because both immobilization and dissociation of the protein can be achieved under physiological conditions, thereby maintaining the full biolog- ical activity of the proteins. [4] Moreover, this approach should also open up ways to restore functional proteins from nanoparticle probes subsequent to their use in bioanalytical assays. To experimentally investigate our hypothesis, we chose DNA conjugates of enhanced yellow fluorescent protein (EYFP), a mutant derivative of the naturally occurring green fluorescent protein from the North Pacific jellyfish Aequorea victoria, as a model system. This choice was made because the DNA–EYFP conjugate is considered to be a promising building block for the construction of biomimetic antennae and light-harvesting devices. [16,17] We also expected it to be a convenient reporter because the fluorescence of the EYFP should be quenched upon binding to the gold nanoparticles, as previously reported for organic fluorescent dyes. [18–24] Initial experiments were carried out to investigate the binding of the covalent DNA–EYFP conjugate (1 in Figure 1, EYFP-5’-AGC GGA TAA CAA TTT CAC ACA GGA-3’) with DNA-coated 25-nm AuNPs 2–4, which contain either complementary (2 : AuNP-5’-TCC TGT GTG AAA TTG TTA TCC GCT-3’, 3 : AuNP-3’-TCG CCT ATT GTT AAA GTG TGT CCT-5’) or, as a control, noncomplementary DNA oligomers (4 : AuNP-5’-TTT TTT TTT TTT GAT CCA GTA GAT A-3’) as specific binding sites. Gel electrophoretic analysis was used to confirm the specific binding (Figure 1). In the case of DNA–AuNPs bearing complementary oligomers, which lead to conjugates 1·2 and 1·3, a clear decrease in the electrophoretic mobility of the nanoparticles indicated the binding of the DNA–EYFP conjugate. In contrast, incubation of particles 4 with 1 only led to very small shifts of the respective bands (Figure 1d, lanes J–L), thus confirming that the binding of 1 with 2 and 3 occurs primarily as a result of specific Watson–Crick base pairing of the oligonucleotides. [25] We then investigated whether the binding of 1 to the gold nanoparticle does indeed lead to quenching of the EYFP fluorescence (Figure 2). Fluorescence measurements were carried out with a solution of 1 (1.2 pmol in TETBS (Tris-HCl Figure 1. DNA–AuNPs 2 and 3 functionalized with EYFP conjugate 1 to form hybrid conjugates a) 1·2 and b) 1·3, along with c) free conjugate 1 and noncomplementary DNA–AuNP 4. d) Electrophoretic analysis of functionalization of gold nanoparticles with EYFP on a 1.5% agarose gel. The bands indicate the mobility of 2 (lane A), 2 coupled with 16, 32, and 64 molar equivalents of 1 (lanes B–D, respectively), 3 (lane E), 3 coupled with 16, 32, and 64 molar equiv- alents of 1 (lanes F–H, respectively), 4 (lane I), and 4 mixed with 16, 32, and 64 molar equivalents of 1 (lanes J–L, respectively). [*] Dr. P. Hazarika, Dipl. Chem. F. Kukolka, Prof. Dr. C. M. Niemeyer Biologisch–Chemische Mikrostrukturtechnik Fachbereich Chemie Universität Dortmund Otto-Hahn Strasse 6, 44227 Dortmund (Germany) Fax: (+ 49)231-755-7082 E-mail: christof.niemeyer@uni-dortmund.de [**] This work was supported in part by Deutsche Forschungsgemein- schaft (DFG, grants Ni-399/6-1/6-2) and by the Zentrum für Angewandte Chemische Genomik, a joint research initiative founded by the European Union and the Ministry of Innovation and Research of the state of North Rhine-Westfalia. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Angewandte Chemie 6827 Angew. Chem. Int. Ed. 2006, 45, 6827 –6830 # 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim