Preprints
https://doi.org/10.5194/mr-2021-58
https://doi.org/10.5194/mr-2021-58

  20 Jul 2021

20 Jul 2021

Review status: this preprint is currently under review for the journal MR.

Rapid measurement of Watson-Crick to Hoogsteen exchange in unlabeled DNA duplexes using high-power SELOPE imino 1H CEST

Bei Liu1, Atul Rangadurai1, Honglue Shi2, and Hashim Al-Hashimi1,2 Bei Liu et al.
  • 1Department of Biochemistry, Duke University School of Medicine, Durham, NC, USA
  • 2Department of Chemistry, Duke University, Durham, NC, USA

Abstract. In duplex DNA, Watson-Crick A-T and G-C base pairs (bps) 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 understanding the role of these non-canonical bps in DNA recognition and repair. However, such studies are hampered by the need to prepare 13C or 15N isotopically enriched DNA samples for NMR relaxation dispersion (RD) experiments. Here, using SELective Optimized Proton Experiments (SELOPE) 1H CEST experiments employing high-power radiofrequency fields (B1 > 250 Hz) targeting imino protons, we demonstrate accurate and robust characterization of Waston-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 1H CEST were in very good agreement with counterparts measured using off-resonance 13C/15N spin relaxation in the rotating frame (R). It is shown that 1H-1H NOE effects which typically introduce artifacts in 1H based measurements of chemical exchange can be effectively suppressed by selective excitation, provided that the relaxation delay is short (≤ 100 ms). The 1H CEST experiment can be performed with ~10X higher throughput and ~100X lower cost relative to 13C/15N R, and enabled Hoogsteen chemical exchange measurements undetectable by R. The results reveal an increased propensity to form Hoogsteen bps near terminal ends and a diminished propensity within A-tract motifs. The 1H CEST experiment opens the door to more comprehensively characterizing Hoogsteen breathing in duplex DNA.

Bei Liu et al.

Status: open (until 17 Aug 2021)

Comment types: AC – author | RC – referee | CC – community | EC – editor | CEC – chief editor | : Report abuse
  • RC1: 'Comment on mr-2021-58', Anonymous Referee #1, 02 Aug 2021 reply
  • RC2: 'Comment on mr-2021-58', Anonymous Referee #2, 05 Aug 2021 reply

Bei Liu et al.

Viewed

Total article views: 246 (including HTML, PDF, and XML)
HTML PDF XML Total Supplement BibTeX EndNote
191 53 2 246 28 2 1
  • HTML: 191
  • PDF: 53
  • XML: 2
  • Total: 246
  • Supplement: 28
  • BibTeX: 2
  • EndNote: 1
Views and downloads (calculated since 20 Jul 2021)
Cumulative views and downloads (calculated since 20 Jul 2021)

Viewed (geographical distribution)

Total article views: 239 (including HTML, PDF, and XML) Thereof 239 with geography defined and 0 with unknown origin.
Country # Views %
  • 1
1
 
 
 
 
Latest update: 05 Aug 2021
Download
Short summary
There is growing interest in mapping exchange dynamics between Watson-Crick and Hoogsteen conformations across different DNA contexts. However, current methods are ill-suited for measurements at a large scale because they require isotopically enriched samples. We report that Hoogsteen dynamics can be measured on unlabeled samples using 1H CEST experiments, which have higher throughput and lower cost relative to conventional methods and also provide new insights into Hoogsteen dynamics.