Detection of Epigenetically Important 5-Formylcytosine Modifications Using Novel Photoreactive Oligonucleotides...

Transcription is tightly regulated by epigenetic modifications that control the expression of specific genes. One such modification is 5-methylcytosine (5mC) that is formed by cytosine methylation via DNA methyltransferases and functions as a transcriptional silencer of gene promoters. Dynamically regulated subsequent modifications by ten-eleven translocation (TET) enzymes catalyze the sequential oxidation of 5mC for generating 5-hydroxymethylcytosine, 5-formylcytosine (5fC), and 5-carboxylcytosine. Recently, these oxidized cytosine derivatives were indicated as not merely intermediates in the demethylation process; however, each derivative plays a unique biologically relevant role. A photo-crosslinkable oligonucleotide probe for detecting these cytosine modifications could support the spatiotemporal functional analysis of cytosine derivatives via a light stimulus. Herein, we designed novel photoreactive nucleosides by conjugating a psoralen (Ps) derivative, trioxsalen, to the C2' position of guanosine (GPs and GPs2) and developed photo-crosslinkable oligonucleotides (GPs-Oligo and GPs2-Oligo). In particular, GPs-Oligo demonstrated an intriguing and unique ability of photo-crosslinking with 5fC. GPs-Oligo was employed to detect 5fC using a DNA chip glass plate, which demonstrated the potential of GPs-Oligo for future investigation of the function of 5fC inside cells.