The visual detection of specific double-stranded DNA sequences possesses great potential for 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 �-lactamase (SEER-LAC), a system consisting of two inactive fragments of �-lactamase each linked to engineered ZFPs. Here the SEER-LAC system was applied to develop a ZFP array that can function as a simple and portable device to identify bacterial double-stranded DNA sequences.  The SEER-LAC system was compared with a detection method with a full length enzyme linked to ZFPs.       As an initial proof of concept, ZFPs were designed to detect a non-pathogenic strain of E. coli. Three pairs of ZFPs were engineered to detect 16S rDNA sequence (rrsA) which is only found in bacteria.  A simple two-probe complementation assay was constructed to detect specific DNA sequences.  One sequence-specific ZFP probe [ZFP(A)] was immobilized on the hydrogel-coated surface.  Double-stranded DNA fragments and a second ZFP probe [ZFP(B)] were then applied to the surface sequentially.  Each probe would carry an inactive fragment of the enzyme TEM-1 �-lactamase, LacA and LacB. Enzyme reassembly would only occur when both target sequences were present in the correct spacing and orientation. Enzyme activity was measured in a final step, using the colorimetric substrate nitrocefin, which produced a signal that is both quantitative and visual. This system therefore provides an isothermic enzymatic amplification of a visual signal that indicates the presence of a specific, user-defined DNA sequence.      The ZFP array with the SEER-LAC system provided a quantitative assay with a detection limit of 50 fmole of target DNA and sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that the ZFP array has the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens.

Detection of specific DNA sequences utilizing custom designed DNA-binding proteins

248th National Meeting (2014)

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