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Despite many techniques that have been developed to detect and sequence 5m C and 5hm C, including recent advances in base-resolution mapping of 5hm C (16), no method to date can simultaneously reveal 5m C and 5hm C sites in the same DNA molecule. This method is highly modular and can be used to image just one modification or multiple modifications simultaneously.Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5m C and 5hm C from trace samples, which we used to study the distance relationship between 5m C and 5hm C with single-molecule fluorescence resonance energy transfer (sm FRET). To image 5hm C, the DNA fragments are first end-labeled with biotin and Cy3 by using Terminal Transferase (Td T) and modified d CTP.DNA epigenetic modifications in the forms of 5-methylcytosine (5m C) and 5-hydroxylmethylcytosine (5hm C) play crucial regulatory functions in the mammalian genome.Here we developed an ultrasensitive single-molecule epigenetic imaging technology for detecting and quantifying 5hm C and 5m C.The dye-labeled biotinylated DNA is then captured by surface-tethered neutravidin on a passivated microscope slide and imaged with single-molecule total internal reflection fluorescence (TIRF) microscopy (Fig. Using synthetic DNA constructs we confirmed that the stepwise labeling is highly efficient and shows minimum background (Fig. We then applied the method to postnatal day 60 (P60) and postnatal day 14 (P14) mouse cerebellum genomic DNA (Fig. Our results for 5hm C are comparable to what has been found in bulk samples by previous HPLC-MS techniques (19) and also reveal the age-dependent increase of 5hm C in mouse cerebellum from P14 to P60 as previously reported (17).

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Because 5m C is much more abundant than 5hm C, we switched the fluorescence labels on the modification for the sm FRET experiment so that there are more acceptors (5m C-Cy5) than donors (5hm C-Cy3) (Fig. We first used synthetic DNA with 5hm C and 5m C separated by defined distances for sm FRET measurement.DNA fragments are end-labeled with biotin, 5hm C is labeled with Cy5, and 5m C is labeled with Cy3.The labeled DNA is immobilized to the microscope slide and imaged with single-molecule TIRF microscopy. Positive control is synthetic DNA bearing biotin and fluorophore.Thanks to the ultrahigh sensitivity of single-molecule imaging, this method only requires 50 pg of DNA or less for each measurement, representing orders of magnitude less DNA than is required by previous quantification methods such as the HPLC-MS or other fluorescence-based methods (19, 21–23).Besides being a general detection and quantification method, single-molecule imaging provides a unique opportunity to study the colocalization states of 5m C and 5hm C, which has been unknown because no previous method could perform an integrated analysis of 5m C and 5hm C in the same DNA molecule.

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