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. In duplex DNA, Watson–Crick A–T and G–C base pairs (bp's) exist in dynamic equilibrium with an alternative Hoogsteen conformation, which is lowin abundance and short-lived. Measuring how the Hoogsteen dynamics variesacross different DNA sequences, structural contexts and physiologicalconditions is key for identifying potential Hoogsteen hot spots and forunderstanding the potential roles of Hoogsteen base pairs in DNA recognitionand repair. However, such studies are hampered by the need to prepare13C or 15N isotopically enriched DNA samples for NMR relaxationdispersion (RD) experiments. Here, using SELective Optimized ProtonExperiments (SELOPE) 1H CEST experiments employing high-powerradiofrequency fields (B1 &gt; 250 Hz) targeting imino protons,we demonstrate accurate and robust characterization of Watson–Crick toHoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pHconditions, the exchange parameters deduced from high-power imino 1HCEST were in very good agreement with counterparts measured usingoff-resonance 13C / 15N spin relaxation in the rotating frame(R1ρ). It is shown that 1H–1H NOE effects which typicallyintroduce artifacts in 1H-based measurements of chemical exchange canbe effectively suppressed by selective excitation, provided that therelaxation delay is short (≤ 100 ms). The 1H CEST experiment can beperformed with ∼ 10× higher throughput and ∼ 100× lower cost relative to 13C / 15N R1ρ and enabledHoogsteen chemical exchange measurements undetectable by R1ρ. Theresults reveal an increased propensity to form Hoogsteen bp's near terminalends and a diminished propensity within A-tract motifs. The 1H CESTexperiment provides a basis for rapidly screening Hoogsteen breathing induplex DNA, enabling identification of unusual motifs for more in-depthcharacterization.

magnetic resonance

Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino &amp;lt;sup&amp;gt;1&amp;lt;/sup&amp;gt;H CEST