8-Aza-2'-deoxyisoguanosine (4) is the first fluorescent shape mimic of 2'-deoxyisoguanosine (1a); its fluorescence is stronger in alkaline medium than under neutral conditions. Nucleoside 4, which was synthesized from 8-aza-2'-deoxyguanosine via a 4,6-diamino intermediate after selective deamination, was incorporated in oligodeoxyribonucleotides using phosphoramidite 11. Duplexes with 4.m(5)iC(d) (5-methyl-2'-deoxyisocytidine) base pairs are more stable than those incorporating dG-dC pairs, thereby expanding the genetic alphabet by a fluorescent orthogonal base pair. As demonstrated by T-m measurements, the base pair stability decreases in the order m(5)iC(d).4 >> dG.4 > dT.4 >= dC.4 >> dA.4. A better base pairing selectivity of 4 against the canonical nucleosides dT, dC, dA, and dG is observed than for the degenerated base pairing of la. The base pair stability changes can be monitored by nucleobase anion fluorescence sensing. The fluorescence change correlates to the DNA base pair stability. Oligonucleotide 5'-d(T(4)4(4)T(4)) (22), containing short runs of nucleoside 4, forms stable multistranded assemblies (ionophores) with K+ in the central cavity. They are quite stable at elevated temperature but are destroyed at high pH value.