We also considered the reverse structural arrangement containing the imidazolium bridged 2-thiouridine dinucleotide (tUpAIptU) intermediate and two adenosine nucleotides in the complementary position of the templating strand. However, both clustering algorithms returned clusters with very unspecific binding of the tUpAIptU dinucleotide, which either interacts with the sugar phosphate backbone of the major grove of RNA double helix (23.9%) or it partly stacks with the adenine nucleobases at 5 ′ -end of the templating strand (32.3%). The clusters found with the K-means procedures display the O-stacked arrangement of tUpAIptU being dominant, and the canonical arrangement of the dinucleotide was found only for one cluster with 6.9% of population. This latter cluster also involves a single WC base pair between the adjacent tU nucleobase and the complementary adenine nucleobase. Overall, the very low binding specificity of tUpAIptU found in our simulations seems inconsistent with the improved rate and fidelity of nonenzymatic RNA template copying observed experimentally . This result seems somewhat surprising when compared to increased rate and fidelity of nonenzymatic template copying involving activated tU nucleotides. This inconsistency could originate from weaknesses of the nucleic acid force field, particularly since our extended parameter library includes non-canonical phosphoroimidazolide groups and alternative nucleobases, which still require much more thorough benchmarking and testing.