Moon-Soo Kim, and Anu Ganesh Kini. Mol. Cells 2017;40:533-41. https://doi.org/10.14348/molcells.2017.0139

Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (L-ascorbic acid) at the indium–tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification.

Ilchan Song, Young Koung Lee, Jin Wook Kim, Seung-Won Lee, Se Ra Park, Hae Kyung Lee, Soyeon Oh, Kinarm Ko, Mi Kyung Kim, Soon Ju Park, Dae Heon Kim, Moon-Soo Kim, Do Sun Kim, and Kisung Ko. Mol. Cells 2021;44:770-9. https://doi.org/10.14348/molcells.2021.2002

A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 β-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.

As antibiotic resistance has risen as one of the major health concerns associated with infectious diseases due to the reduced efficacy of antibiotics, rapid and sensitive detection of antibiotic resistance genes is critical for more effective and faster treatment of infectious diseases. A class of programmable DNA-binding domains called transcriptional activator-like effectors (TALEs) provides a novel scaffold for designing versatile DNA-binding proteins due to their modularity and predictability. Here, we developed a simple, rapid, and sensitive system for detecting antibiotic resistance genes by exploring the potential of TALE proteins for the creation of a sequence-specific DNA diagnostic along with 2D-nanosheet graphene oxide (GO). TALEs were engineered to directly recognize the specific double-stranded (ds) DNA sequences present in the tetracycline resistance gene (tetM), avoiding the need for dsDNA denaturation and renaturation. We take advantage of the GO as an effective signal quencher to quantum dot (QD)-labeled TALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbed on the GO surface, which will bring QDs in close proximity to GO. Due to the fluorescence quenching ability of GO, QDs are expected to be quenched by GO via fluorescence resonance energy transfer (FRET). QD-labeled TALE binding to the target dsDNA would lead to the conformational change, which would result in dissociation from the GO surface, thereby restoring the fluorescence signal. Our sensing system was able to detect low concentrations of dsDNA sequences in the tetM gene after only 10-minute incubation with the DNA, providing a limit of detection as low as 1 fM of Staphylococcus aureus genomic DNA. This study demonstrated that our approach of utilizing TALEs as a new diagnostic probe along with GO as a sensing platform can provide a highly sensitive and rapid method for direct detection of the antibiotic resistance gene without requiring DNA amplification or labeling.