Base pairing involving artificial bases in vitro and in vivo

Herein we report the synthesis of N(8)-glycosylated 8-aza-deoxyguanosine (N(8)-8-aza-dG) and 8-aza-9-deaza-deoxyguanosine (N(8)-8-aza-9-deaza-dG) nucleotides and their base pairing properties with 5-methyl-isocytosine (d-isoC(Me)), 8-amino-deoxyinosine (8-NH(2)-dI), 1-N-methyl-8-amino-deoxyinosine (1-Me-8-NH(2)-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-isoC(Me):d-isoG artificial genetic system. As demonstrated by T(m) measurements, the N(8)-8-aza-dG:d-isoC(Me) base pair formed less stable duplexes as the C:G and d-isoC(Me):d-isoG pairs. Incorporation of 8-NH(2)-dI versus the N(8)-8-aza-dG nucleoside resulted in a greater reduction in T(m) stability, compared to d-isoC(Me):d-isoG. Insertion of the methyl group at the N(1) position of 8-NH(2)-dI did not affect duplex stability with N(8)-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-NH(2)-dI and 8-NH(2)-dI) and N(8)-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.