Selective and stable base pairing by alkynylated nucleosides featuring a spatially-separated recognition interface

Unnatural base pairs (UBPs) which exhibit a selectivity against pairing with canonical nucleobases provide a powerful tool for the development of nucleic acid-based technologies. As an alternative strategy to the conventional UBP designs, which involve utility of different recognition modes at the Watson-Crick interface, we now report that the exclusive base pairing can be achieved through the spatial separation of recognition units. The design concept was demonstrated with the alkynylated purine (Pu-N, Pu-O) and pyridazine ((N)Pz, (O)Pz) nucleosides endowed with nucleobase-like 2-aminopyrimidine or 2-pyridone ('pseudo-nucleobases') on their major groove side. These alkynylated purines and pyridazines exhibited exclusive and stable pairing properties by the formation of complementary hydrogen bonds between the pseudo-nucleobases in the DNA major groove as revealed by comprehensive T-m measurements, 2D-NMR analyses, and MD simulations. Moreover, the alkynylated purine-pyridazine pairs enabled dramatic stabilization of the DNA duplex upon consecutive incorporation while maintaining a high sequence-specificity. The present study showcases the separation of the recognition interface as a promising strategy for developing new types of UBPs.