The sensitive detection of DNA is of great interest and demand for an increasing number of biomedical studies and diagnostic applications. Zinc Finger (ZF) domains provide a versatile scaffold to construct customized DNA-binding proteins that recognize specific DNA sequences. Novel transcriptional activator-like effector (TALE) domains were recently discovered to have a previously undescribed DNA-binding motif. The development of simple, rapid, and robust technologies for detection of pathogens is becoming critical due to the increasing rate of mortality and risk of bacteremia. PCR-based pathogen detection is a favorable alternative to culture methods, offering sensitive means of detecting multiple pathogens. However, this technology will likely remain expensive and bulky for the widespread dissemination in limited-resource settings. The use of sequence-specific DNA-binding proteins provides a useful scaffold for ‘direct’ reading of the sequence information, avoiding the need for denaturation and subsequent renaturation required by current DNA microarray technology. In addition, ZF and TALE domains can be engineered for recognition of specific sequences in multiple pathogens and arrayed on the surface, which could lead to development of a simple non-PCR based technology for multiplexed detection of pathogens. The next generation TALE DNA-binding based technology could offer the possibility of more sensitive diagnostics, since TALE domains seem to overcome certain limitation of ZFs. Here, engineered ZFPs to detect shiga toxin in E. coli O157:H7 were immobilized on the COC chip, providing a detection limit of 10 fmole of target DNA. When combined with an electrochemical method, the ZFP array obtained 105 fold-improved sensitivity. Our data suggest that the DNA-binding protein array could be an innovative and promising approach for sensitive and sequence-specific detection of multiple pathogens, requiring no DNA labeling and hybridization.

Novel protein array for direct detection of double-stranded DNA sequences for diagnostic applications

250th National Meeting (2015)

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