Supplementary Figure 1: 4-thiouracil-derivatization by thiol-linked alkylation.
From: Thiol-linked alkylation of RNA to assess expression dynamics
(a) 4-thiouracil (s4Uracil) reacts with the thiol-reactive compound iodoacetamide (IAA), attaching a carboxyamidomethyl-group to the thiol-group in s4Uracil because of a nucleophilic substitution (SN2) reaction. Absorption maxima of educt (4-thiouracil; s4Uracil; λmax ≍ 335 nm) and product (carboxyamidomethylated 4-thiouracil; *s4Uracil; λmax ≍ 297 nm) are indicated. (b) Absorption spectra of 4-thiouracil (s4Uracil) in the absence and presence of the indicated concentration of iodoacetamide (IAA). 1 mM s4U was incubated with the indicated concentration of IAA for 1 h at 37˚C in the presence of 50 mM sodium phosphate buffer (pH 8.0) and 10% DMSO. Data represents mean ± SD of independent experiments (untreated n=13; 1mM & 10 mM IAA n=3). (c) Quantification of absorption at 335 nm as shown in (B). P-values (two-tailed Student’s t-test) are indicated. (d) Absorption spectra of 1 mM 4-thiouracil (s4Uracil) in the absence and presence of 10 mM iodoacetamide (IAA) after incubation at the indicated temperature for 5 min in the presence of 50 mM sodium phosphate buffer (pH 8.0) and 10% DMSO. Data represents mean ± SD of independent experiments (untreated n=9; 37˚C, 50˚C, 80˚C, n=3). (e) Quantification of absorption at 335 nm as shown in (d). P-values (two-tailed Student’s t-test) are indicated. (f) Absorption spectra of 1 mM 4-thiouracil (s4Uracil) in the absence and presence of 10 mM iodoacetamide (IAA) after incubation at 37˚C for the indicated time in the presence of 50 mM sodium phosphate buffer (pH 8.0) and 10% DMSO. Data represents mean ± SD of independent experiments (untreated n=15; 5 min, 15 min, 75 min n=3). (g) Quantification of absorption at 335 nm as shown in (f). P-values (two-tailed Student’s t-test) are indicated. (h) Absorption spectra of 1 mM 4-thiouracil (s4Uracil) in the absence and presence of 10 mM iodoacetamide (IAA) after incubation at 50˚C for 2 min in the presence of 50 mM sodium phosphate buffer (pH 8.0) and the indicated amount of DMSO. Data represents mean ± SD of independent experiments (untreated n=13; 10%, 30 %, 50% DMSO n=3). (i) Quantification of absorption at 335 nm as shown in (H). P-values (two-tailed Student’s t-test) are indicated. (j) Absorption spectra of 1 mM 4-thiouracil (s4Uracil) in the absence and presence of 10 mM iodoacetamide (IAA) after incubation at 50˚C for 5 min in the presence of 50 mM sodium phosphate buffer with the indicated pH and the 10 % DMSO. Data represents mean ± SD of independent experiments (untreated n=25; pH6, 7, 8 n=3). (k) Quantification of absorption at 335 nm as shown in (j). P-values (two-tailed Student’s t-test) are indicated. (l) Absorption spectra of 1 mM 4-thiouracil (s4Uracil) in the absence and presence of 10 mM iodoacetamide (IAA) after incubation at 50˚C for 15 min in the presence of 50 mM sodium phosphate buffer (pH 8.0) and 50 % DMSO (optimal reaction [rxn] conditions). Data represent mean ± SD of independent experiments (untreated n=13; optimal reaction conditions n=3). (m) Quantification of absorption at 335 nm as shown in (j). P-values (two-tailed Student’s t-test) are indicated. Points represent individual measurements. Mean (center line) ± SD (whiskers) is indicated.
