The identity of the nucleophile substitution may influence metal interactions with the cleavage site of the minimal hammerhead ribozyme

Abstract

Potential metal interactions with the cleavage site of a minimal hammerhead ribozyme (mHHRz) were probed using (31)P NMR-detected Cd(2+) titration studies of HHRz constructs containing a phosphorothioate (PS) modification at the cleavage site. The mHHRz nucleophile position was replaced by either a 2'-F or a 2'-NH(2) in order to block cleavage activity during the study. The 2'-F/PS cleavage site mHHRz construct, in which the 2'-F should closely imitate the atom size and electronegativity of a 2'-OH, demonstrates low levels of metal ion association (<1 ppm (31)P chemical shift changes). This observation indicates that having an atom size and electrostatic properties that are similar to the 2'-OH are not the governing factors in allowing metal interactions with the scissile phosphate of the mHHRz. With a 2'-NH(2) substitution, a large upfield change in (31)P NMR chemical shift of the phosphorothioate peak (Delta approximately 3 ppm with 6 equiv of added Cd(2+)) indicates observable Cd(2+) interactions with the substituted site. Since a 2'-NH(2), but not a 2'-F, can serve as a metal ligand, these data suggest that a metal ion interaction with the HHRz cleavage site may include both the scissile phosphate and the 2' nucleophile. Control samples in which the 2'-NH(2)/PS unit is placed either next to the mHHRz cleavage site (at U16.1), in a duplex, or in a (am)U(PS)U dinucleotide show much weaker interactions with Cd(2+). Results with these control samples indicate that simply the presence of a 2'-NH(2)/PS unit does not create a strong metal binding site, reinforcing the possibility that the 2'-NH(2)-moderated Cd-PS interaction is specific to the mHHRz cleavage site. Upfield chemical shifts of both (31)P and H-2' (1)H resonances in (am)U(PS)U are observed with addition of Cd(2+), consistent with the predicted metal coordination to both 2'-NH(2) and phosphorothioate ligands. These data suggest that metal ion association with the HHRz cleavage site may include an interaction with the 2'-OH nucleophile.

Figure 1

(A) Sequence of truncated HHRz (trHHRz) used in this study. Nucleotides in blue identify the substrate strand, and green nucleotides represent the enzyme strand. The conserved core is shown in red, and an arrow identifies the site of cleavage (cleavage site, CS) between nucleotides C17 and G1.1. (B) 2′-nucleophile substitutions used in this study, in combination with phosphorothioate substitutions. (C) X-ray crystal structure of an all-RNA mHHRz (PDB1MME) (48) showing location of the phosphodiester groups 3′ to C17 (CS) and U16.1 examined in this study using ³¹P NMR spectroscopy.

Figure 2

Cd²⁺-induced phosphorothioate ³¹P chemical shift changes in the mHHRz and control samples. (A) R_p or S_p PS substitutions 5′ to A9 (blue, red) or at the cleavage site (CS) with a 2′-OMe nucleophile substitution (yellow, green), and in a control duplex (purple, gold). Data reproduced from Maderia et al. 2000 (17)). (B) R_p or Sp PS substitutions in the HHRz with a 2′-NH₂ substitution (2′NH₂/PS) at the cleavage site (CS, red and blue) or at U16.1 (green and pink). Also shown are data for a control duplex (2′NH₂/PS) (purple and gold), and a dinucleotide ^amU_PSU (brown and green). All RNA concentrations are ~300–500 μM.

Figure 3

³¹P NMR spectra of HHRz with a R_p or S_p 2′-F/PS substitution at the cleavage site. Experiments were performed in 5mM HEPES (pH 8.5) and 100 mM Na⁺ at 15 °C.

Figure 4

³¹P NMR of HHRz samples with 2′-NH₂/PS substitution (mixture of PS diastereomers) at the cleavage site (C17, left) or the 5′ neighboring position (U16.1, right). Data were obtained at 15°C in 5 mM HEPES (pH 8.5) and 100 mM NaCl with initial RNA concentrations of 450 μM (left) and 440 uM (right).

Figure 5

DQF-COSY spectra of ^amU_SpU in absence (black) and presence (red) of 40 mM CdCl₂. Data were obtained in D₂O at 10 °C in 10 mM sodium cacodylate (pH 7.4) and 100 mM NaCl. Crosspeak assignments are shown for the 2′NH₂ ribose system (solid lines) and partial assignment for the 2′OH ribose (dotted line).

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