Oligonucleotides incorporating the unusually linked 8-aza-7-deazapurine N⁸-(2′-deoxyribonucleosides) 3a,b (purine and 6-amino-2-chloropurine analogues) were used as chemical probes to investigate the base pairing motifs of the universal nucleoside 8-aza-7-deazapurin-6-amine N⁸-(2′-deoxyribofuranoside) 2 (adenine analogue) and that of the 2,6-diamino compound 1. Owing to the absence of an amino group on the nucleoside 3a the low stability of oligonucleotide duplexes incorporating this compound opposite to the four canonical DNA-constituents indicate hydrogen bonding and base pairing for the universal nucleosides 1 and 2 which form much more stable duplexes. When the 6-amino-2-chloro-8-aza-7-deazapurine nucleoside 3b replaces 1 and is located at the same positions, two sets of duplexes are formed (i) high T_m duplexes with 3b located opposite to dA or dC and (ii) low T_m duplexes with 3b located opposite to dG or dT. These results are due to the steric clash of the 2-chloro substituent of 3b with the 2-oxo group of dT or the 6-oxo group of dG while the 2-halogeno substituents are well accommodated in the base pairs formed with dA or dC. For comparison duplexes incorporating the regularly linked nucleosides 4–6a,b containing the same nucleobases as those of 1–3a,b were studied.