The development of new tools for tracking the activity of human DNA methyltransferases is an important goal given the role of this enzyme as a cancer biomarker and epigenetic modulator. However, analysis of the human DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity is challenging, especially in crude samples, because of the low activity and large size of the enzyme. Here, we report a new approach to Dnmt analysis that combines nanostructured electrodes with a digest-and-amplify strategy that directly monitors Dnmt1 activity with high sensitivity. Nanostructured electrodes are required for the function of the assay to promote the accessibility of the electrode for human Dnmt1. Moreover, DNA-templated deposition of silver nanoparticles (for signal amplification) is combined with DNA Exonuclease I digestion to yield optimal target-to-control signals. We achieve high sensitivity for the detection of human Dnmt1, and particularly Dnmt1 from crude cell lysates. Specifically, the detection limit of our electrochemical assay is 20 pM, which is 2 orders of magnitude lower than previously reported methods. In crude lysates, we detected Dnmt1 from as few as five colorectal cancer cells (HCT116). With biopsy samples, we were able to distinguish colorectal tumor tissue from healthy adjacent tissue using only 10 μg of sample. The strategy enables analysis of an important marker underlying the epigenetic basis of cancerous transformation.

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http://dx.doi.org/10.1021/acssensors.8b00494DOI Listing

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