For comparison, we also considered the opposite configuration with the imidazolium bridged cytosine dinucleotide intermediate (CpAIpC) and two complementary guanosine nucleotides in the templating strand. The DBSCAN analysis returned a single cluster with the population of 99.9% (Fig. )). As demonstrated through the representative frame, the adjacent cytosine nucleobase is positioned at the binding site, forming WC hydrogen bonds with the complementary guanine nucleobase. However, the terminal cytidine adopted a non-canonical orientation (an O-stacked structure, see below), which maximized the stacking between the two cytosine nucleobases of CpAIpC. Consequently, the terminal nucleotide of CpAIpC interacts with the complementary guanine through two hydrogen bonds: one connecting the N-H group of guanine with the carbonyl oxygen of cytosine, and the other one connecting the 2 ′ -OH group of cytidine with the carbonyl group of the guanine nucleobase on the template. Both nucleobases of CpAIpC adopted the anti conformation, with the adjacent cytidine having a C2 ′ -endo conformation of the sugar, while the terminal nucleotide was in C3 ′ -endo conformation. It is important to emphasize, though, that this frame presented in Fig. ) does not perfectly represent all of the structures included in this cluster. Consequently, the K-means allowed us to better discriminate between the different arrangements of CpAIpC bound to the template, which particular emphasis on the terminal nucleotide. In particular, we found similar structures in the O-stacked geometry, and related conformers having the templating terminal guanosine nucleotide rotated further away from the CpAIpC dinucleotide (see Supporting Information Fig. ). Additionally, K-means identified two clusters, representing 31.4% of all the analysed frames, which closely resemble the crystal structure of GpAIpG (Fig. )) and maintain WC interactions as well as. In these clusters, the ribose moieties are predominantly in the C2 ′ -endo conformation, with the exception of the terminal nucleobases in one cluster, along with the anti orientation of cytosine We next considered two systems with mixed activated dinucleotide intermediates which consisted of one cytidine and guanosine monomer, namely CpAIpG and GpAIpC. The former one contained cytidine as the adjacent nucleotide, while GpAIpC was oriented in the opposite direction on the template. In both cases we included the complementary WC nucleotides on the templating strand. For CpAIpG, K-means algorithm identified clusters with structurally similar arrangements, from which three prominent structural patterns can be distinguished (Fig. )-f)). The major pattern populated in 41.8% resembles canonically stacked GpAIpG along with anti configuration of nucleobases and WC hydrogen bonding interactions with the complementary nucleobases on the templating strand. In this case, the dominant ribose conformation within the CpAIpG dinucleotide is C3 ′ -endo. Another structural pattern populated in 32.7%, which can be seen for two other clusters from the K-means analysis involves the terminal activated guanosine nucleotide displaced sideways from the templating strand, with no stacking interactions between the two terminal nucleotides within the template. The final structural motif populated in 21.5% is characterised by the nucleotide at 5 ′ terminus of the templating strand acting as a loose overhang and the activated guanosine nucleotide of CpAIpG taking its place in the stacking interaction with the second to the last guanosine nucleotide of the template. Importantly, in all of these clusters the activated adjacent cytidine nucleotide of CpAIpG maintains a WC base pairing interaction with its partner on the template.