Molecular imaging of aging-related 5-hydroxymethylcytosine in cell-free DNA at the single-copy level

In cell-free DNA (cfDNA), 5-hydroxymethylcytosine (5hmC) is a vital epigenetic modification closely related to aging. Although many methods have been developed to quantify 5hmC in DNA extracted from cells, few have been successfully applied to detect 5hmC in cfDNA due to their insufficient sensitivity. In our work, we quantified the ratio of 5hmC modified cfDNA in the circulating blood of volunteers at different ages using an ultrasensitive single-molecule immunofluorescent imaging method. Among the results, the ratio of 5hmC modified cfDNA in the old volunteers was significantly higher than that in the young volunteers. In addition, the sensitivity of this method was greatly improved with the minimal input of 0.5 pg cfDNA, compared to the traditional dot blot assay. Moreover, old volunteers with geriatric syndromes exhibited a significantly higher level of 5hmC in cfDNA than their counterparts without such syndromes. Last, in a single-molecule photobleaching experiment, we demonstrated that the higher ratio of 5hmC modified cfDNA among the old volunteers may result from the enrichment of 5hmC in a single cfDNA strand. Taken together, 5hmC in circulating cfDNA may play multiple roles in aging-related physical changes, especially in geriatric syndromes, and could be a biomarker for evaluating such syndromes.