Base pairing involving artificial bases in vitro and in vivo

Herein we report the synthesis of N8‐glycosylated 8‐aza‐deoxyguanosine (N8‐8‐aza‐dG) and 8‐aza‐9‐deaza‐deoxyguanosine (N8‐8‐aza‐9‐deaza‐dG) nucleotides and their base pairing properties with 5‐methyl‐isocytosine (d‐isoCMe), 8‐aminodeoxyinosine (8‐NH2‐dI), 1‐N‐methyl‐8‐amino‐deoxyinosine (1‐Me‐8‐NH2‐dI), 7,8‐dihydro‐8‐oxo‐deoxyinosine (8‐Oxo‐dI), 7,8‐dihydro‐8‐oxo‐deoxyadenosine (8‐Oxo‐dA), and 7,8‐dihydro‐8‐oxo‐deoxyguanosine (8‐Oxo‐dG), in comparison with the d‐isoCMe:d‐isoG artificial genetic system. As demonstrated by Tm measurements, the N8‐8‐aza‐dG:d‐isoCMe base pair formed less stable duplexes as the C:G and d‐isoCMe:d‐isoG pairs. Incorporation of 8‐NH2‐dI versus the N8‐8‐aza‐dG nucleoside resulted in a greater reduction in Tm stability, compared to d‐isoCMe:d‐isoG. Insertion of the methyl group at the N1 position of 8‐NH2‐dI did not affect duplex stability with N8‐8‐aza‐dG, thus suggesting that the base paring takes place through Hoogsteen base pairing. The cellular interpretation of the nucleosides was studied, whereby a lack of recognition or mispairing of the incorporated nucleotides with the canonical DNA bases indicated the extent of orthogonality in vivo. The most biologically orthogonal nucleosides identified included the 8‐amino‐deoxyinosines (1‐Me‐8‐NH2‐dI and 8‐NH2‐dI) and N8‐8‐aza‐9‐deaza‐dG. The 8‐Oxo modifications mimic oxidative damage ahead of cancer development, and the impact of the MutM mediated recognition of these 8‐oxo‐deoxynucleosides was studied, finding no significant impact in their in vivo assay.status: publishe