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Rapid assessment of Watson–Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino (1)H CEST
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 conditi...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Copernicus GmbH
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10539785/ https://www.ncbi.nlm.nih.gov/pubmed/37905209 http://dx.doi.org/10.5194/mr-2-715-2021 |
Sumario: | 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 [Formula: see text] C or [Formula: see text] N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) [Formula: see text] H CEST experiments employing high-power radiofrequency fields ([Formula: see text] [Formula: see text] 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 [Formula: see text] H CEST were in very good agreement with counterparts measured using off-resonance [Formula: see text] C [Formula: see text] [Formula: see text] N spin relaxation in the rotating frame ([Formula: see text]). It is shown that [Formula: see text] H– [Formula: see text] H NOE effects which typically introduce artifacts in [Formula: see text] H-based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short ([Formula: see text] 100 ms). The [Formula: see text] H CEST experiment can be performed with [Formula: see text] 10 [Formula: see text] higher throughput and [Formula: see text] 100 [Formula: see text] lower cost relative to [Formula: see text] C [Formula: see text] [Formula: see text] N [Formula: see text] and enabled Hoogsteen chemical exchange measurements undetectable by [Formula: see text]. The results reveal an increased propensity to form Hoogsteen bp's near terminal ends and a diminished propensity within A-tract motifs. The [Formula: see text] H CEST experiment provides a basis for rapidly screening Hoogsteen breathing in duplex DNA, enabling identification of unusual motifs for more in-depth characterization. |
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