Probing Transient Hoogsteen Hydrogen Bonds in Canonical Duplex DNA Using NMR Relaxation Dispersion and Single-Atom Substitution

Abstract

Nucleic acids transiently morph into alternative conformations that can be difficult to characterize at the atomic level by conventional methods because they exist for too little time and in too little abundance. We recently reported evidence for transient Hoogsteen base pairs in canonical B-DNA based on NMR carbon relaxation dispersion. While the carbon chemical shifts measured for the transient state were consistent with a syn orientation for the purine base, as expected for A(syn)•T(anti) and G(syn)•C+(anti) HG base pairing, HG type hydrogen bonding could only be inferred indirectly. Here, we develop two independent approaches for directly probing transient changes in N-H⋯N hydrogen bonds and apply them to the characterization of transient Hoogsteen type hydrogen bonds in canonical duplex DNA. The first approach takes advantage of the strong dependence of the imino nitrogen chemical shift on hydrogen bonding and involves measurement of R1ρ relaxation dispersion for the hydrogen-bond donor imino nitrogens in G and T residues. In the second approach, we assess the consequence of substituting the hydrogen-bond acceptor nitrogen (N7) with a carbon (C7H7) on both carbon and nitrogen relaxation dispersion data. Together, these data allow us to obtain direct evidence for transient Hoogsteen base pairs that are stabilized by N-H⋯N type hydrogen bonds in canonical duplex DNA. The methods introduced here greatly expand the utility of NMR in the structural characterization of transient states in nucleic acids.

Widespread transient Hoogsteen base pairs in canonical duplex DNA with variable energetics

“…A two-state analysis $false(\mathrm{normalA}\underset{k_B}{\overset{k_A}{\rightleftarrows }}\mathrm{normalB}false)$ of the R 1ρ data 4–6 (see Methods ) yielded populations ( p B = ~0.08–2.73 %) and lifetimes (τ B = ~0.12–2.57 ms) for the transient state (Supplementary Fig. 2 and Supplementary Table 2) that are similar to those reported previously for transient HG bps ( p B = ~0.14–0.49 % and τ B = ~0.3–2.5 ms) 1,2 . The chemical shifts (ω B ) of the transient state obtained using this analysis (Supplementary Fig.…”

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

“…The methyl group eliminates one Watson-Crick hydrogen-bond as well as sterically collides with the partner pyrimidine effectively destabilizing the Watson-Crick bp without significantly impacting the Hoogsteen conformation (Figure 1B). The chemical shifts of the m 1 A + -T and m 1 G-C + Hoogsteen bps recapitulate those measured for the ES using NMR R1ρ in unmodified duplexes, indicating that they are valid models for the ES Hoogsteen bps 10,41,45,46 .…”

“…For example, the T9-C3’ is predicted to experience active RD due to formation of A16-T9 Hoogsteen bp and remote RD due to formation of a neighboring G10-C + 15 Hoogsteen bp. Since the active and remote chemical shift perturbations are opposite (Figure 3C and 3D), and since Hoogsteen transitions at different sites occur independently 10,46 , this site is predicted to experience 3-state exchange in an RD experiment. While other sites such as A17-C1′, A17-C8, and G10-C3′ are also predicted to experience active and remote RD, the perturbations have similar signs, and the contribution from active RD is predicted to dominate (see Supplementary Discussion).…”

“…These include the 13 C 10,45 and 15 N chemical shifts 46 , the enhanced populations seen with inosine substitutions 73 , diminished populations observed with 7-deazopurine substitutions 46 , and the pH dependence of the exchange process 69 . The strong agreement observed here between the Δω values measured for the base and sugar moieties of the transient A-T and G-C + Hoogsteen bps as well as their neighbors with values measured in the Hoogsteen stabilizing mutants in two different duplexes, as well as the strong agreement between the measured Δω values and those predicted using AFQM/MM leave little doubt that the ESs measured in duplex DNA correspond to Hoogsteen bps.…”

Nuclear spin relaxation in liquids and gases

“…Nikolova et al 130 described two methods for probing transient Hoogsteen type N-H?? ?N hydrogen bonds in canonical duplex DNA.…”