An A‐T Hoogsteen Base Pair in a Naked DNA Hairpin Motif: A Protein‐Recognized Conformation

In duplex DNA, A‐T and G‐C form Watson‐Crick base pairs, and Hoogsteen pairing only dominates upon protein binding or DNA damage. Using NMR, we show that an A‐T Hoogsteen base pair previously observed in crystal structures of transposon DNA hairpins bound to TnpA protein forms in solution even in the absence of TnpA. This Hoogsteen base pair, located adjacent to a dinucleotide apical loop, exists in dynamic equilibrium with a minor Watson‐Crick conformation (population ~11% and lifetime ~55 µs). Extending the apical loop to three residues inverted the equilibrium, making Watson‐Crick the dominant state and the Hoogsteen conformation recognized by TnpA a minor state (population ~14% and lifetime ~28 µs). The propensity for Hoogsteen pairing depended on apical loop residues, which form contacts directly or indirectly stabilizing the Hoogsteen conformation. A structure survey did not reveal Hoogsteen pairing near RNA apical loops making them unique to DNA. Our results demonstrate that Hoogsteen can be the dominant state even in naked unmodified duplex DNA and identify 5’‐CTT(T/C)AG‐3’ as a DNA‐specific apical loop motif stabilized by Hoogsteen pairing. Hoogsteen conformations may be prevalent in DNA hairpins forming during replication and transcription, with broad implications for the genomic landscape.