A zinc finger protein array for the visual detection of specific DNA sequences for diagnostic applications Moon-Soo Kim 1 , Gulnaz Stybayeva 2 , Ji Youn Lee 2 , Alexander Revzin 2 and David J. Segal 1, * 1 Genome Center, Department of Pharmacology and 2 Department of Biomedical Engineering, 451 Health Sciences Drive, University of California, Davis, CA 95616, USA Received July 22, 2010; Revised November 6, 2010; Accepted November 10, 2010 ABSTRACT The visual detection of specific double-stranded DNA sequences possesses great potential for the development of diagnostics. Zinc finger domains provide a powerful scaffold for creating custom DNA-binding proteins that recognize specific DNA sequences. We previously demonstrated sequence- enabled reassembly of TEM-1 b-lactamase (SEER– LAC), a system consisting of two inactive fragments of b-lactamase each linked to engineered zinc finger proteins (ZFPs). Here the SEER–LAC system was applied to develop ZFP arrays that function as simple devices to identify bacterial double-stranded DNA sequences. The ZFP arrays provided a quanti- tative assay with a detection limit of 50 fmol of target DNA. The method could distinguish target DNA from non-target DNA within 5 min. The ZFP arrays provided sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that ZFP arrays have the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens. INTRODUCTION Sensitive and specific detection of DNA is of great interest and demand for an increasing number of biomedical studies and applications, including clinical diagnostics. The development of simple, robust and rapid technologies for the detection of pathogens is particularly crucial, considering the high rate of mortality associated with bloodborne infection (1). The availability of portable devices for the multiplexed detection of pathogens in blood would also be highly significant for disaster management, as well as treatment of patients in resource- limited settings. For pathogen detection, lab-on-chip technologies are well suited for point-of-care (POC) diag- nostics. Incorporating molecular diagnostics on a microfluidic technology would facilitate the development of POC device. Polymerase chain reaction (PCR)-based methods have been established as quick and sensitive methods for the detection of pathogens, in contrast to laborious culture methods (2,3). However, diagnostic situations often require multiplexed pathogen detection (4,5). Multiplex PCR can identify several genes in one reaction. On the other hand, DNA microarrays offer the capability to detect a much larger number of pathogens simultaneously. To take advantage of this capability, the combination of PCR with microarray detection has been studied (6–10). Most of the detection methods are based on DNA hybrid- ization with single-stranded DNA probes (6,11–13). However, DNA hybridization is time consuming, and duplex formation can be affected by the formation of sec- ondary structures in the probes (6,12,13). In contrast, sequence specific DNA-binding proteins read the sequence information directly from double-stranded DNA, avoiding the need for denaturation and subsequent renaturation with probes under controlled conditions. A common type of DNA-binding domain is the Cys2- His2 zinc finger, which contains about 30 amino acids (14). The Cys2-His2 zinc finger domain provides a versa- tile scaffold to construct customized DNA-binding proteins that specifically recognize virtually any desired DNA sequence (14,15). The amino acids of the zinc finger form a bba fold, which is stabilized by hydrophobic interactions and a zinc ion coordinated by the two conserved cysteine and histidine residues. Each finger typ- ically recognizes three to four nucleotides of DNA. Zinc finger domains can be linked into tandem arrays that allow mutifinger proteins to recognize extended DNA *To whom correspondence should be addressed. Tel: +530 754 9134; Fax:+530 754 9658; Email: djsegal@ucdavis.edu Published online 5 December 2010 Nucleic Acids Research, 2011, Vol. 39, No. 5 e29 doi:10.1093/nar/gkq1214 ß The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.