A single-point substitution of the O4' oxygen by a CH₂ group at the sugar residue of A⁶ (i.e. 2'-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5'-d(C¹G²C³G⁴A⁵A⁶T⁷T⁸C⁹ G¹⁰C¹¹G¹²)₂^-3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A⁶:T⁷ Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)↔(1B): K_eq = k₁/k_-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k₁ (298K) = 3.9±0.8 sec ^-1; ΔH °^‡ = 164±14 kJ/mol; -TΔS °^‡ (298K) = -92 kJ/mol giving a ΔG °^‡ ₂₉₈ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0 ±0.6 sec-1, ΔH °^‡ = 153±13 kJ/mol; -TΔS °^‡ (298K) = -82 kJ/mol giving a ΔG °^‡ ₂₉₈ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of ΔG °^‡ ₂₉₈ of the forward (k1) and backward (k-1) conversions, (1A)↔(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A⁶:T⁷ Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A⁶:T⁷ basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2'-deoxyaristeromycin moiety, A⁶, as a result of substitution of the endocyclic 4'-oxygen in the natural sugar with a methylene group in A⁶. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.