Thiones

All RNA oligomers were synthesized on an ABI 394 Synthesizer (Life Technologies, Carlsbad, CA, USA) using 2′-O-thionocarbamate-protected nucleoside phosphoramidites (Sigma-Aldrich, St. Louis, MO, USA or Thermo Fisher, Waltham, MA, USA) according to previously described procedures^{6 (link)}. 2′-O-methyl phosphoramidites were purchased from Thermo Scientific, Grand Island, NY, and incorporated into RNA oligomers under the same conditions as the 2′-O-thionocarbamate protected phosphoramidites. The 2′-O-methyl-3′-O-(diisopropylamino)phosphinoacetic acid-1,1-dimethylcyanoethyl ester-5′-O-dimethoxytrityl nucleosides used for synthesis of thiophosphonoacetate (thioPACE)-modified RNAs were synthesized essentially according to published methods^{9 (link),22 (link)}. For phosphorothioate containing oligomers, the iodine oxidation step after the coupling reaction was replaced by a sulfurization step using a 0.05 M solution of 3-((N,N-dimethylamino methylidene)amino)-3H-1,2,4-dithiazole-5-thione in a pyridine-acetonitrile (3:2) mixture for 6 min. Unless noted otherwise, reagents for solid phase RNA synthesis were purchased from Glen Research (Sterling, VA, USA).
All oligonucleotides were purified using reversed phase high-performance liquid chromatography (HPLC) and analyzed by liquid chromatography– mass spectrometry (LC-MS) using an Agilent 1290 Infinity series LC system coupled to an Agilent 6520 Q-TOF (time-of-flight) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The yields for the synthesis and purification of the sgRNAs were estimated using deconvolution of mass spectra obtained from LC-MS-derived total ion chromatograms. The chemical synthesis of the 100-mer sgRNAs typically yielded 25–35% full-length product from a nominal 1 micromole scale synthesis. Reversed-phase HPLC purification using ion pairing buffer conditions typically gave 20% yield from the crude product with an estimated purity of the final sgRNA in the range of 90% to 95%. Supplementary Table 1 shows the sequences of all sgRNAs used and the masses obtained from deconvolution of the multiple charge state series of peaks found from analysis of the purified sgRNAs. The deconvolution was done using Mass Hunter Qualitative Analysis (version B.06.00) software (Agilent).

A solution of 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol; 200 mg, 1.41 mmol) in HCl (0.38 M, 2 mL) in a 10 mL microwave reaction vessel was treated with amine (3.1 mmol, 2.2 equiv). The heterogenous reaction mixture was sealed and stirred at 65 °C for 5 min prior to one microwave cycle of 1.5 min at 165 °C, 250 psi (max P), and 300 W. The reaction typically produced a yellow solution with a black, oily residue at the bottom of the tube. The reaction mixture was extracted with CH₂Cl₂ (2×) and the organic phase was dried (MgSO₄), vacuum filtered and evaporated in vacuo to give a dark residue. Purification by silica column chromatography (CH₂Cl₂ or 0–2 % MeOH in CH₂Cl₂) gave the desired product.