Abstract:Until recently,i tw as believed that the genomes of higher organisms contain, in addition to the four canonical DNAb ases,o nly 5-methyl-dC (m 5 dC) as am odified base to control epigenetic processes.I nr ecent years,t his view has changed dramatically with the discovery of 5-hydroxymethyldC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNAfrom stem cells and brain tissue.N 6 -methyldeoxyadenosine (m 6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms.T his base,t ogether with N 4 -methyldeoxycytidine (m 4 dC), was first reported to be ac omponent of bacterial genomes.I nt his work, we investigated the levels and distribution of these potentially epigenetically relevant DNAb ases by using an ovel ultrasensitive UHPLC-MS method. We further report quantitative data for m 5 dC,h mdC,f dC,a nd cadC,b ut we were unable to detect either m 4 dC or m 6 dA in DNAisolated from mouse embryonic stem cells or brain and liver tissue,w hichc alls into question their epigenetic relevance.The genetic material of living organisms is constructed from the four canonical nucleobases dA, dC,d G, and dT,w hich establish the sequence information that, in multicellular organisms,i ss tored in the nucleus of every cell ( Figure 1). In addition to the canonical bases,the methylated dC base 5-methyldeoxycytidine (m 5 dC) is frequently found.[1] The presence or absence of this base in specific promoter segments determines whether the gene is actively transcribed or silenced.[1] Thec ell-type-specific distribution of m 5 dC thus determines the identity of ag iven cell. Recently,5 -hydroxymethyldeoxycytidine (hmdC) was found as as ixth base of the genetic system [2,3] and in 2011, 5-formyldeoxycytidine (fdC) [4,5] and 5-carboxydeoxycytidine (cadC) [5,6] were also discovered, particularly in DNAisolated from stem cells,but also in brain DNA. It is currently believed that fdC and cadC are intermediates in an active demethylation process that allows cells to change the methylation pattern and hence the activity state of specific genes. [7,8] ForfdC,separate epigenetic functions are also envisaged. [9] While the genomes of bacteria are known to also contain N 4 -methyldeoxycytidine (m 4 dC) [10] and N 6 -methyldeoxyadenosine (m 6 dA), [11] attempts to detect these bases in the DNA of higher organisms have failed until recently. [12][13][14][15] m 6 dA has now been found in algae (0.4 mol %m 6 dA/A), [12] fruit flies (0.001 %-0.07 %m 6 dA/A), [14] and C. elegans (0.01 %-0.4 % m 6 dA/A), [13] and its presence has even been reported in the DNAo fv ertebrates (0.00009 %i nX. laevis [16] and 0.00019-0.003 %o fd Ai nm urine cells and tissue [17] ). These discoveries,e specially concerning the DNAo fv ertebrates,h ave spurred aw orldwide research interest in unraveling the function of these new bases in human genomic DNA. [18][19][20] In this study,w ed eveloped an ultrasensitive triple quadrupole mass spectrometry (QQQ-MS) method, which in combination with ultra-high-pressure chromatograph...

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