To compare the above systems to activated dinucleotide intermediates which were suggested as weaker binders, we next investigated the binding of activated uridine dinucleotide intermediate (UpAIpU) to adenosine nucleotides on the template containing complementary adenosine residues. The structure of the UpAIpU dinucleotide representing the highest populated cluster generated by the DBSCAN algorithm (68.3% of the population) involves only one WC base-pairing interaction which is formed between the adjacent uracil of UpAIpU and the complementary adenine (see Fig. )). The terminal uracil of UpAIpU is oriented away from the interaction site and forms the so-called T-shape interaction with the complementary adenine nucleobase of the template, where the terminal uracil is arranged in a perpendicular manner with respect to it . Both uracil nucleobases exhibited the anti orientation with respect to the ribose moieties, with terminal nucleotide in the C2 ′ -endo conformation and the adjacent adopting the C3 ′ -endo conformation. In the second highest cluster from the DBSCAN analysis (28.8% of population), and also four clusters from the K-means analysis (total population of 29.9%), the UpAIpU dinucleotide diffused away from the binding site and interacted with the sugar phosphate backbone on the opposite side of the RNA double helix (Supporting Information Fig. and). Importantly, the K-means clustering procedure revealed that even though the UpAIpU dinucleotide can be found near the interaction site for ∼69% of the time, its structure tends to largely sample random coil arrangements or O-stacked structures. Reasonable recognition for at least one A and U pair was found for merely 14.5% of the simulation frames (Supporting Information Fig. and).