Harnessing Alternative Substrates to Probe TET Family Enzymes.

TET family enzymes normally oxidize 5-methylcytosine (5mC) in DNA, and play critical roles in shaping the epigenome. Despite their importance, assessing TET activity can be difficult, particularly given the challenge of studying modifications to single nucleobases within complex DNA substrates. We recently demonstrated that in addition to acting on 5mC, TET enzymes can act promiscuously on unnatural nucleobases. Here, we describe how these alternative unnatural substrates can be employed in facile assays to detect and quantify TET activity. DNA containing unnatural 5-vinylcytosine (vC) can be used as a direct endpoint reporter of TET activity, a method that can potentially be adapted to high-throughput platforms. Complementarily, DNA containing unnatural 5-ethynylcytosine (eyC) can trap and inactivate TET enzymes upon reaction, a strategy that can be used to extract active TET enzymes from a complex cellular milieu. We present a detailed PCR-based protocol to synthesize DNA probes with either natural or unnatural modifications, and methods for using these probes to track TET activity either in vitro or in cell extracts.