1H‐ and 31P‐nuclear magnetic resonance studies on the conformation of d‐(CpGpCpG)2 and d‐(CpGpCpGpCpG)2 short helices in B‐conformation

Abstract

AbstractTwo short DNA helices, d‐(CpGpCpG)2 and d‐(CpGpCpGpCpG)2, in dilute salt solutions have been studied thoroughly by 1H‐ and 31P‐nmr spectroscopy as model systems for the calibration of nmr theory in its application to nucleic acids research. All the nonexchangeable proton resonances of these two short helices have been assigned by the “incremental method,” “sequential homodecoupling” of the sugar protons, thermal perturbation, and computer spectral simulation, as well as the NOE technique.The CD and uv data were also obtained at either identical or half concentrations of the oligomer used in the nmr studies in order to have comparable optical data for estimating helical content and for assigning conformation. The precise assignment of the sugar proton resonances and their corresponding coupling‐constant values provide valuable information about the backbone conformation. The sugar conformation was shown to be predominantly in the 2E‐form (>70%). The rotation of the C4′C5′ is in favor of the gg conformation (55–95%), the rotation of the C5′O5′ bond is highly in favor of g′g′ (83–91%), and the rotation of the C3′O3′ bond is restricted to the domain of ϕ′ ≃ 195°.The CD data and the sugar conformational analyses indicate that these CG helices are in the B‐form in dilute salt solutions. The assignment of NHN hydrogen‐bonded resonances of these two helices are based on their thermal stability. The through‐space magnetic field effects on the base protons and the NHN protons in the helices were calculated and compared with the experimental values. This comparison yields two general conclusions: (1) For the base protons, application of both ring‐current effects and local anisotropic effects in the computations are preferred; but for the NHN protons, the application of the ring‐current effect alone is preferred over the application of both ring‐current effects and local anisotropic effects; and (2) while the computed values in the best theoretical treatment cited above did support the conclusion that these CG helices are in the B‐conformation, the differences among the computed values for the three conformations, A, A′, and B, are not very large, at least for the CG sequence. All the 31P resonances of these CG helices are unambiguously assigned through a specific heterodecoupling technique established previously [Cheng et al. (1982) Biopolymers 21, 697–701]. The complete assignment of all proton resonances and 31P resonance of d‐(CpGpCpG)2 and d‐(CpGpCpGpCpG)2 helices in the B‐form establishes these two deoxy CG helices as valuable model systems for the study of the nmr and polymorphism of nucleic acids.