Influence of nucleotide identity on ribose 2'-hydroxyl reactivity in RNA

Abstract

Hydroxyl-selective electrophiles, including N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitroisatoic anhydride (1M7), are broadly useful for RNA structure analysis because they react preferentially with the ribose 2'-OH group at conformationally unconstrained or flexible nucleotides. Each nucleotide in an RNA has the potential to form an adduct with these reagents to yield a comprehensive, nucleotide-resolution, view of RNA structure. However, it is possible that factors other than local structure modulate reactivity. To evaluate the influence of base identity on the intrinsic reactivity of each nucleotide, we analyze NMIA and 1M7 reactivity using four distinct RNAs, under both native and denaturing conditions. We show that guanosine and adenosine residues have identical intrinsic 2'-hydroxyl reactivities at pH 8.0 and are 1.4 and 1.7 times more reactive than uridine and cytidine, respectively. These subtle, but statistically significant, differences do not impact the ability of selective 2'-hydroxyl acylation analyzed by primer extension-based (SHAPE) methods to establish an RNA secondary structure or monitor RNA folding in solution because base-specific influences are much smaller than the reactivity differences between paired and unpaired nucleotides.

FIGURE 1.

Reaction of electrophiles with the 2′-hydroxyl position in RNA. (A) Scheme for SHAPE chemistry. (B,C) Secondary structures and normalized SHAPE reactivities analyzed under native and denaturing conditions. A representative region of 16S rRNA is shown.

FIGURE 2.

Box plot analysis of SHAPE reactivities for the entire denatured RNA data set. Upper and lower panels illustrate experiments performed with NMIA and 1M7. Equalities at the bottom left of each group emphasize nucleotides showing statistically equivalent reactivities. Boxes outline the middle 50% of each data set; medians are indicated with bold lines. Whiskers above and below each box give the largest or smallest nonoutlier values; outliers are indicated by open circles and are >1.5 times the interquartile range (box).

FIGURE 3.

Differential reactivity of unpaired (un) and internally (int) paired nucleotides toward NMIA and 1M7. Paired nucleotides react within a tighter range and have a smaller mean reactivity than do unpaired nucleotides.

FIGURE 4.

Box plots of nucleotides that are single-stranded in natively folded RNAs. Reactivities toward NMIA and 1M7 are shown in the upper and lower panels, respectively. Statistical equalities are indicated at the lower left of each plot.

FIGURE 5.

Nucleotide-specific reactivities for NMIA and 1M7. Reactivities for NMIA and 1M7 are reported as the mean plus an error term (root-mean square of the coefficient of variation). These values are compared with other reagents that form stable covalent adducts with RNA. Numerical estimates for the relative nucleotide-specific reactivity of each reagent were obtained from: DMS (Lawley and Shah 1972), bisulfite and kethoxal (Ehresmann et al. 1987), DEPC (Peattie 1979), and CMCT (Gilham 1962).