Protonation of nucleobases in anions of canonical 2'-deoxyribonucleotides has been investigated by the DFT computational study at the B3LYP/aug-cc-pvdz level of theory. It is demonstrated that the protonation leads to a significant decrease of conformational space of purine nucleotides while almost all conformers found for non-protonated molecules correspond to minima of the potential energy surface for protonated mdTMP and mdCMP. However, in all nucleotides, only one conformer is populated. This applies to all tautomers of protonated molecules except the mdTMP and mdCMP with the proton attached to the carbonyl group where a minor population of second conformer is observed Protonation of nucleobase leads to significant elongation of the N-glycosidic bond. These findings agree well with suggestions that protonation of nucleobase is a first step in cleavage of the glycosidic bond The oxygen atoms of both carbonyl groups of thymine and the N3 atom of the pyrimidine ring of cytosine, guanine, and adenine represent the most preferable sites for protonation of anions of 2'-deoxyrobonudeotides. The highest proton affinity is observed for the base in mdGMP and the lowest for the thymine moiety in mdTMP. It should be noted that calculated values of the proton affinities in anionic nucleotides are significantly higher (by 2-3 eV) than for nucleosides and neutral nucleotides. This allows assuming that the proton affinity of the base in DNA macromolecule may be tuned by changing the extent of shielding or neutralization of negative charge of the phosphate group.