The solution structure of a DNA*RNA duplex containing 5-propynyl U and C; comparison with 5-Me modifications

Abstract

The addition of the propynyl group at the 5 position of pyrimidine nucleotides is highly stabilising. We have determined the thermodynamic stability of the DNA.RNA hybrid r(GAAGAGAAGC)*d(GC(p)U(p)U(p)C(p)U(p) C(p)U(p)U(p)C) where p is the propynyl group at the 5 position and compared it with that of the unmodified duplex and the effects of methyl substitutions. The incorporation of the propyne group at the 5 position gives rise to a very large stabilisation of the hybrid duplex compared with the analogous 5-Me modification. The duplexes have been characterised by gel electrophoresis and NMR spectroscopy, which indicate that methyl substitutions have a smaller influence on local and global conformation than the propynyl groups. The increased NMR spectral dispersion of the propyne-modified duplex allowed a larger number of experimental restraints to be measured. Restrained molecular dynamics in a fully solvated system showed that the propyne modification leads to substantial conformational rearrangements stabilising a more A-like structure. The propynyl groups occupy a large part of the major groove and make favourable van der Waals interactions with their nearest neighbours and the atoms of the rings. This enhanced overlap may account at least in part for the increased thermodynamic stability. Furthermore, the simulations show a spine of hydration in the major groove as well as in the minor groove involving the RNA hydroxyl groups.

Figure 1

NMR spectra of the propyne-modified DNA·RNA hybrid duplex. Spectra were recorded at 303 K and 600 MHz as described in the Materials and Methods. (A) NOESY spectrum recorded with a mixing time of 250 ms. The base-H1′ region is shown, with connectivities of the DNA and RNA strands. Red, RNA purine strand; cyan, modified DNA pyrimidine strand; green, Ade H2 intra and cross-strand NOEs. (B) Double quantum filtered COSY spectrum of the H1′ to H2′/H2” region showing the coupling patterns for the deoxyriboses. The acquisition time in t₂ was 0.68 s.

Figure 1

NMR spectra of the propyne-modified DNA·RNA hybrid duplex. Spectra were recorded at 303 K and 600 MHz as described in the Materials and Methods. (A) NOESY spectrum recorded with a mixing time of 250 ms. The base-H1′ region is shown, with connectivities of the DNA and RNA strands. Red, RNA purine strand; cyan, modified DNA pyrimidine strand; green, Ade H2 intra and cross-strand NOEs. (B) Double quantum filtered COSY spectrum of the H1′ to H2′/H2” region showing the coupling patterns for the deoxyriboses. The acquisition time in t₂ was 0.68 s.

Figure 2

Chemical shift differences between modified and unmodified duplexes. Chemical shift differences were obtained for non-exchangeable protons at 30°C using the unmodified duplexes as the reference as described in the text. Open bars, H8/H6; closed bars, H1′; shaded bars, H2′; horizontal striped bars, H3”. (A) dR10·r^MeY10–dR10·rY10. (B) rR10·dY10– rR10·dY(U)10. (C) rR10·d^pY10–rR10·dY(U)10.

Figure 3

Structures of the propyne-modified hybrid duplex compared with dY·rR. Averaged structures are displayed looking into the major groove and down the helix axis. Left, rR·dY [from Gyi et al. (9)]. Right, rR·d^pY. The propyne groups are shown in yellow.

Figure 3

Structures of the propyne-modified hybrid duplex compared with dY·rR. Averaged structures are displayed looking into the major groove and down the helix axis. Left, rR·dY [from Gyi et al. (9)]. Right, rR·d^pY. The propyne groups are shown in yellow.

Figure 4

Hydration rR·d^pY. Hydration density was calculated as described in the Materials and Methods. The white contours for water are drawn at 6-fold greater than average water density. (A) Minor groove hydration. (B) Hydration in the 5-propyne:pyrimidine phosphate backbone groove. (C) Major groove hydration.