Standard reagents

Oligonucleotides (ONs) were synthesized using a Biosset ASM-800 DNA synthesizer (Biosset Ltd., Russia) and standard reagents (Glen Research, USA) following standard phosphoramidite protocols. Modified ONs were synthesized using standard reagents and 5′-O-dimethoxytrityl-1′,2′-dideoxyribose-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (GlenResearch, USA). ONs were purified by a preparative-scale reverse-phase high-performance liquid chromatography (HPLC) on a 250 mm × 4.0 mm Hypersil C18 column (Thermo Fisher Scientific, USA) with detection at λ = 260 nm and a 2.5–15% gradient of acetonitrile in 0.1 M ammonium acetate buffer at 55°C. Dimethoxytrityl protection groups were removed via treatment with 80% acetic acid for 30 min, and the detritylated ONs were further purified in a 2.5–15% gradient of acetonitrile in 0.1 M ammonium acetate buffer. The purity of all ONs was approximately 95% by HPLC. The ON concentrations were calculated from the absorbance measured above 90°C and extinction coefficients, which were approximated using the nearest-neighbor model.

Oligonucleotides were synthesized in-house on UnyLinker CPG solid supports (ChemGenes), using a MerMade 12 synthesizer (BioAutomation). Standard reagents (Glen Research) and coupling procedures were used, with acetonitrilic 4,5-dicyanoimidazole as the activator and iodine as the oxidizer. For sequences with amine modifications, amino-modifier C6 dT (#10-1039, Glen Research) was used at the specified positions (table S1).
Oligonucleotides were cleaved from the solid support using standard AMA deprotection [1:1 mixture of 30% NH₄OH (aq) and 40% methylamine (aq) for 25 min at 55°C] and purified using reversed-phase high-performance liquid chromatography (HPLC) (Agilent 1260 Infinity) equipped with an Agilent Dynamax Microsorb C18 column, with a gradient of 0 to 75% acetonitrile in triethylammonium acetate buffer over 45 min. With collected fractions, dimethoxytrityl protecting groups were cleaved under 20% (v/v) acetic acid for 1 hour and lyophilized overnight. The identities of the purified oligonucleotides were confirmed by matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) using a Bruker MALDI Rapiflex Tissue Typer in linear negative mode, with 2′,6′-dihydroxyacetophenone as the matrix and diammonium hydrogen citrate as the co-matrix.