Journal of Biomolecular NMR

, Volume 70, Issue 4, pp 229–244 | Cite as

Atomic structures of excited state A–T Hoogsteen base pairs in duplex DNA by combining NMR relaxation dispersion, mutagenesis, and chemical shift calculations

  • Honglue Shi
  • Mary C. Clay
  • Atul Rangadurai
  • Bharathwaj Sathyamoorthy
  • David A. Case
  • Hashim M. Al-HashimiEmail author


NMR relaxation dispersion studies indicate that in canonical duplex DNA, Watson–Crick base pairs (bps) exist in dynamic equilibrium with short-lived low abundance excited state Hoogsteen bps. N1-methylated adenine (m1A) and guanine (m1G) are naturally occurring forms of damage that stabilize Hoogsteen bps in duplex DNA. NMR dynamic ensembles of DNA duplexes with m1A–T Hoogsteen bps reveal significant changes in sugar pucker and backbone angles in and around the Hoogsteen bp, as well as kinking of the duplex towards the major groove. Whether these structural changes also occur upon forming excited state Hoogsteen bps in unmodified duplexes remains to be established because prior relaxation dispersion probes provided limited information regarding the sugar-backbone conformation. Here, we demonstrate measurements of C3′ and C4′ spin relaxation in the rotating frame (R1ρ) in uniformly 13C/15N labeled DNA as sensitive probes of the sugar-backbone conformation in DNA excited states. The chemical shifts, combined with structure-based predictions using an automated fragmentation quantum mechanics/molecular mechanics method, show that the dynamic ensemble of DNA duplexes containing m1A–T Hoogsteen bps accurately model the excited state Hoogsteen conformation in two different sequence contexts. Formation of excited state A–T Hoogsteen bps is accompanied by changes in sugar-backbone conformation that allow the flipped syn adenine to form hydrogen-bonds with its partner thymine and this in turn results in overall kinking of the DNA toward the major groove. Results support the assignment of Hoogsteen bps as the excited state observed in canonical duplex DNA, provide an atomic view of DNA dynamics linked to formation of Hoogsteen bps, and lay the groundwork for a potentially general strategy for solving structures of nucleic acid excited states.


DNA dynamics Rotating frame spin relaxation Nucleic acids Sugar pucker Major groove kinking DNA bending 



We thank all Al-Hashimi lab members for critical comments on the manuscript. We acknowledge the technical support and resources from the Duke Magnetic Resonance Spectroscopy Center. This work was supported by US National Institute for General Medical Sciences (5P50GM103297 to H.M.A. and D.A.C and R01GM089846 to H.M.A.). The authors declare no conflict of interest.

Supplementary material

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Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Honglue Shi
    • 1
  • Mary C. Clay
    • 2
  • Atul Rangadurai
    • 2
  • Bharathwaj Sathyamoorthy
    • 1
    • 2
    • 4
  • David A. Case
    • 3
  • Hashim M. Al-Hashimi
    • 1
    • 2
    Email author
  1. 1.Department of ChemistryDuke UniversityDurhamUSA
  2. 2.Department of BiochemistryDuke University School of MedicineDurhamUSA
  3. 3.Department of Chemistry and Chemical BiologyRutgers UniversityPiscatawayUSA
  4. 4.Department of ChemistryIndian Institute of Science Education and Research BhopalBhopalIndia

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