To evaluate the binding of activated dinucleotide intermediates containing alternative nucleobase we next turned to systems containing inosine (I) and 2-thiouridine (2-tU) nucleotides. We started the investigation with the system containing an imidazolium-bridged inosine dinucleotide intermediate (IpAIpI) interacting with two cytidine nucleotides at the 5 ′ -end of the templating strand. The highest populated cluster generated by DBSCAN analysis (55.5%) is associated with a structure that closely resembles the canonically stacked GpAIpG dinucleotide (Fig. )). Consequently, both inosine residues exhibit a canonical Watson-Crick basepairing interaction, anti conformation of the nucleobases and C3 ′ -endo conformation of the ribose fragments. This canonical structure is also represented by 2 clusters from the K-means procedure with the overall population of 22.8% (see Supporting Information Fig. ). In addition, we see a single related cluster charaterised by a less specific interaction formed between the terminal hypoxanthine (inosine) and cytosine nucleobases, with the WC base pair retained for the adjacent inosine residue (14.4%). However, a considerable proportion of clusters from the K-means procedure (27.7% of the trajectory) are characterized by nonspecific interactions of the backbone of IpAIpI with the complementary strand which do not allow for selective recognition of the activated inosine nucleotides and result in higher distances of the adjacent phosphate group of IpAIpI from the C3 ′ terminus of the primer. The latter feature was suggested to effectively hinder the primer extension reaction . Given that, experimentally activated inosine nucleotides were observed to undergo practically as efficient primer extension reactions as guanosine nucleotides, the observed lower population of the reactive conformation for IpAIpI, requires further analysis and considerations. Importantly, the clustering analysis based on the K-means algorithm revealed another structural arrangements of the IpAIpI dinucleotide (Fig. and)), which includes the hypoxanthine nucleobases folded into one another. We denoted one of these as G-stacked, as its structure resembles the shape the capital letter G (Fig. a)) while the other is termed O-stacked (Fig. )). The Gand O-stacked arrangements of IpAIpI are usually associated with the formation of a single WC base pair for the adjacent inosine nucleotide.