Abstract
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 low in abundance and short-lived. Measuring how the Hoogsteen dynamics varies across different DNA sequences, structural contexts and physiological conditions is key for identifying potential Hoogsteen hot spots and for understanding the potential roles of Hoogsteen base pairs in DNA recognition and repair. However, such studies are hampered by the need to prepare C or N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) H CEST experiments employing high-power radiofrequency fields ( 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Watson-Crick to Hoogsteen exchange, without the need for isotopic enrichment of the DNA. For 13 residues in three DNA duplexes under different temperature and pH conditions, the exchange parameters deduced from high-power imino H CEST were in very good agreement with counterparts measured using off-resonance C N spin relaxation in the rotating frame (). It is shown that H-H NOE effects which typically introduce artifacts in H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short ( 100 ms). The H CEST experiment can be performed with 10 higher throughput and 100 lower cost relative to C N and enabled Hoogsteen chemical exchange measurements undetectable by . The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization.
Highlights
Soon after the discovery of the DNA double helix, it was shown that A–T and G–C could pair in an alternative conformation known as the “Hoogsteen” base pair (Felsenfeld et al, 1957; Hoogsteen, 1959) (Fig. 1a)
We used 1H chemical exchange saturation transfer (CEST) rather than R1ρ given the greater ease of collecting profiles for many spins simultaneously, and given that with the use of high-power RF fields, CEST can effectively characterize exchange processes over a wide range of timescales (Rangadurai et al, 2020a)
Building on prior studies showing the utility of the SELective Optimized Proton Experiments (SELOPE) 1H relaxation dispersion (RD) experiment in measuring conformational exchange in unlabeled RNA (Schlagnitweit et al, 2018) and DNA (Furukawa et al, 2021; Dubini et al, 2020), our study establishes the utility of high-power 1H CEST SELOPE as a facile means for rapidly assessing the Watson–Crick to Hoogsteen exchange process in nucleic acids without the need for isotopic enrichment
Summary
Soon after the discovery of the DNA double helix, it was shown that A–T and G–C could pair in an alternative conformation known as the “Hoogsteen” base pair (bp) (Felsenfeld et al, 1957; Hoogsteen, 1959) (Fig. 1a). Starting from a canonical Watson–Crick G–C or A–T bp, the corresponding Hoogsteen bp’s can be obtained by flipping the purine base 180◦ and bringing the two bases into proximity to create a new set of hydrogen bonds, which in the case of G–C bp’s require protonation of cytosine N3 (Fig. 1a). Following their discovery, Hoogsteen bp’s were observed in crystal structures of duplex DNA in complex with proteins (Kitayner et al, 2010; Aishima et al, 2002) and drugs (Wang et al, 1984; Ughetto et al, 1985) and shown to play a role in DNA recognition (Golovenko et al, 2018), damage induction (Xu et al, 2020), and repair (Lu et al, 2010), and in damage bypass during replication (Nair et al, 2006; Ling et al, 2003). This pulse sequence is adapted from Schlagnitweit et al (2018)
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