Abstract
Natural, covalently modified cytosine bases within genomic DNA function as important epigenetic markers. Approaches for single-base-resolution sequencing of cytosine modifications typically deploy chemistry for modification-selective C-to-T code conversion and can require error-prone subtractive analysis of complex data. Here we report the sequencing of an epigenetic base by exploiting an unnatural base pair system. This approach relies on hydrogen-bonding complementarity between a malononitrile adduct of 5-formylcytosine and protonated 3,7-dideazaadenine. The specificity of this unnatural base pair was studied by biophysical DNA thermal melting analysis and by template-directed incorporation by DNA polymerase enzymes. Base pair selectivity was enhanced by controlling the protonation state of 3,7-dideazaadenine. We exemplify use of this unnatural base pair to sequence 5-formylcytosine in a DNA template using a Sanger-type format. There is scope for this base pair and the general concept to be implemented on further sequencing platforms that exploit Watson-Crick base pairing to directly identify epigenetic bases.