Abstract 5-Formylcytosine (5fC) is a chemically edited, naturally occurring nucleobase which appears in the context of modified DNA strands. The understanding of the impact of 5fC on dsDNA physical properties is to date limited. In this work, we applied temperature-dependent 1H Chemical Exchange Saturation Transfer (CEST) NMR experiments to non-invasively and site-specifically measure the thermodynamic and kinetic influence of formylated cytosine nucleobase on the melting process involving dsDNA. Incorporation of 5fC within symmetrically positioned CpG sites destabilizes the whole dsDNA structure—as witnessed from the ∼2°C decrease in the melting temperature and 5–10 kJ mol−1 decrease in ΔG°—and affects the kinetic rates of association and dissociation. We observed an up to ∼5-fold enhancement of the dsDNA dissociation and an up to ∼3-fold reduction in ssDNA association rate constants, over multiple temperatures and for several proton reporters. Eyring and van’t Hoff analysis proved that the destabilization is not localized, instead all base-pairs are affected and the transition states resembles the single-stranded conformation. These results advance our knowledge about the role of 5fC as a semi-permanent epigenetic modification and assist in the understanding of its interactions with reader proteins.

The removal of 5‐methyl‐deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5‐hydroxymethyl‐deoxycytidine (hmdC) and further on to 5‐formyl‐deoxycytidine (fdC) and 5‐carboxy‐deoxycytidine (cadC) with the help of α‐ketoglutarate‐dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC. A second pathway is proposed to involve C−C bond cleavage that converts fdC directly into dC. A 6‐aza‐5‐formyl‐deoxycytidine (a‐fdC) probe molecule was synthesized and fed to various somatic cell lines and induced mouse embryonic stem cells, together with a 2′‐fluorinated fdC analogue (F‐fdC). While deformylation of F‐fdC was clearly observed in vivo, it did not occur with a‐fdC, thus suggesting that the C−C bond‐cleaving deformylation is initiated by nucleophilic activation.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNAHis, tRNAAsn, tRNATyr, and tRNAAsp. It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNATyr and tRNAAsp of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl‐queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q‐family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q‐nucleoside family.

5-Formyl-deoxyuridine (fdU) and 5-formyl-deoxycytidine (fdC) are formyl-containing nucleosides that are created by oxidative stress in differentiated cells. While fdU is almost exclusively an oxidative stress lesion formed from deoxythymidine (T), the situation for fdC is more complex. Next to formation as an oxidative lesion, it is particularly abundant in stem cells, where it is more frequently formed in an epigenetically important oxidation reaction performed by α-ketoglutarate dependent TET enzymes from 5-methyldeoxycytidine (mdC). Recently, it was shown that genomic fdC and fdU can react with the ɛ-aminogroups of nucleosomal lysines to give Schiff base adducts that covalently link nucleosomes to genomic DNA. Here, we show that fdU features a significantly higher reactivity towards lysine side chains compared with fdC. This result shows that depending on the amounts of fdC and fdU, oxidative stress may have a bigger impact on nucleosome binding than epigenetics.

5‐Methyl‐2′‐Desoxycytidin (mdC) wird als fünfte Base des genetischen Systems betrachtet, dessen Anwesenheit zur Unterdrückung der Expression korrespondierender Gene beiträgt. Die Entfernung der Modifikation und Reaktivierung der unterdrückten Gene ist durch einen noch nicht vollständig aufgeklärten aktiven Demethylierungsmechanismus möglich. Dieser setzt die Oxidationsreaktionen von mdC zum 5‐Hydroxymethyl‐2′‐Desoxycytidin (hmdC) und weiter zum 5‐Formyl‐2′‐Desoxycytidin (fdC) und 5‐Carboxy‐2′‐Desoxycytidin (cadC) voraus, die durch α‐Ketoglutarat‐abhängige Tet‐Oxygenasen katalysiert werden. Die Entfernung von mdC geschieht auf den Oxidationsstufen des fdC und cadC. Neben dem Eingreifen bestimmter Glykosylasen (TDG) beschreibt ein zweiter Weg eine C‐C‐Bindungsspaltung, die fdC direkt in dC umwandelt. Während der TDG‐induzierte Reparaturmechanismus gut charakterisiert ist, existieren zum Mechanismus durch C‐C‐Bindungsbruch noch sehr wenige Informationen. Hier führen wir ein neuartiges 6‐Aza‐5‐Formyl‐2′‐Desoxycytidin‐Testmolekül (a‐fdC) ein, das verschiedenen somatischen Zelllinien und induzierten mESCs zusammen mit einem 2′‐fluorierten fdC‐Analogon (F‐fdC) gefüttert wurde. Während die Deformylierung von F‐fdC in vivo eindeutig beobachtet werden konnte, wurde sie für a‐fdC hingegen nicht beobachtet, was vermuten lässt, dass die Deformylierung über C‐C‐Bindungsbruch nach vorhergehender nukleophiler Aktivierung erfolgt.