NMR analyses on N-hydroxymethylated nucleobases - implications for formaldehyde toxicity and nucleic acid demethylases.

Formaldehyde is produced in cells by enzyme-catalysed demethylation reactions, including those occurring on N-methylated nucleic acids. Formaldehyde reacts with nucleobases to form N-hydroxymethylated adducts that may contribute to its toxicity/carcinogenicity when added exogenously, but the chemistry of these reactions has been incompletely defined. We report NMR studies on the reactions of formaldehyde with canonical/modified nucleobases. The results reveal that hydroxymethyl hemiaminals on endocyclic nitrogens, as observed with thymidine and uridine monophosphates, are faster to form than equivalent hemiaminals on exocyclic nitrogens; however, the exocyclic adducts, as formed with adenine, guanine and cytosine, are more stable in solution. Nucleic acid demethylase (FTO)-catalysed hydroxylation of (6-methyl)adenosine results in (6-hydroxymethyl)adenosine as the major observed product; by contrast no evidence for a stable 3-hydroxymethyl adduct was accrued with FTO-catalysed oxidation of (3-methyl)thymidine. Collectively, our results imply N-hydroxymethyled adducts of nucleic acid bases, formed either by reactions with formaldehyde or via demethylase catalysis, have substantially different stabilities, with some being sufficiently stable to have functional roles in disease or the regulation of nucleic acid/nucleobase activity.

Stable end-sealed DNA as robust nano-rulers for in vivo single-molecule fluorescence.

Single-molecule fluorescence and Förster resonance energy transfer (smFRET) are important tools for studying molecular heterogeneity, cellular organization, and protein structure in living cells. However, in vivo smFRET studies are still very challenging, and a standardized approach for robust in vivo smFRET measurements is still missing. Here, we synthesized protected DNAs with chemically linked ends as robust in vivo nano-rulers. We efficiently internalized doubly-labeled end-sealed DNA standards into live bacteria using electroporation and obtained stable and long-lasting smFRET signatures. Single-molecule fluorescence signals could be extended to ∼1 min by studying multi-fluorophore DNA standards. The high stability of protected DNA standards offers a general approach to evaluate single-molecule fluorescence and FRET signals, autofluorescence background, and fluorophore density, and hence, quality check the workflow for studying single-molecule trajectories and conformational dynamics of biomolecules in vivo.