Role of Pseudoisocytidine Tautomerization in Triplex-Forming Oligonucleotides: In Silico and in Vitro Studies

[Image: see text] Pseudoisocytidine ((Ψ)C) is a synthetic cytidine analogue that can target DNA duplex to form parallel triplex at neutral pH. Pseudoisocytidine has mainly two tautomers, of which only one is favorable for triplex formation. In this study, we investigated the effect of sequence on (Ψ)C tautomerization using λ-dynamics simulation, which takes into account transitions between states. We also performed in vitro binding experiments with sequences containing (Ψ)C and furthermore characterized the structure of the formed triplex using molecular dynamics simulation. We found that the neighboring methylated or protonated cytidine promotes the formation of the favorable tautomer, whereas the neighboring thymine or locked nucleic acid has a poor effect, and consecutive (Ψ)C has a negative influence. The deleterious effect of consecutive (Ψ)C in a triplex formation was confirmed using in vitro binding experiments. Our findings contribute to improving the design of (Ψ)C-containing triplex-forming oligonucleotides directed to target G-rich DNA sequences.