Spectroscopic studies on the structure of poly(8-bromoadenylic acid): effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

Abstract

The helix-coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5'-mono- and polynucleotides. The chemical shifts of H(2'), H(3'), C(2'), and C(3') nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3') signal and a downfield shift of the H(3') signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3') endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2') and H(2') signals when the preferred conformation is C(2')endo-syn (e.g., in 5'-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ⇔ anti and N ⇔ S equilibria in solutions. The three-bond ¹H-'¹³C coupling constants between H(1') and C(8) and C(4) have been measured in poly(8BrA) and 5'-8BrAMP and their structural implications have been discussed. The observed preference of a C(3')endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2')endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^-1. The antisymmetric >PO₂^- stretching vibration is observed at an unusually low frequency in the helix (1214 cm^-1). The symmetric >PO stretch occurs at ~1095 cm^-1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^-1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.