In addition, we performed simulations involving the substitution of uridine nucleotides with 2-tU both for the dinucleotide intermediate and the template. The system containing the ApAIpA dinucleotide with two tU nucleotides on the template exhibited much more specific binding than its counterpart containing biological uridine nucleotides within the template. In particular, we identified two clusters characterised by WC base pairing interactions for both components of ApAIpA with the total population of 19.4%, based on the K-means procedure (Fig. )). Similar to the parent system containing the ApAIpA dinucleotide, the tU modification of the template still resulted in substantial population of G-stacked structures (77.6%), part of which also involved a single WC base-pairing interaction of the adjacent A nucleobase (18.5%). Qualitatively, the results demonstrate that substituting the oxygen atom in the 2 position with a sulfur atom stabilizes the ApAIpA dinucleotide on the template and improves the specificity. This observation is consistent with the experimental data .