Oligonucleotides synthesis

The synthesis of oligonucleotides was performed via MerMade 12 solid phase synthesis with phosphoramidites. All oligonucleotides (DNA, modified DNA, 2'OMeRNA, 2'OMeRNA-LNA) were deprotected according to published procedure^49,50. To solid support was added 32% ammonia solution and incubated in 55 °C, 18 h, than evaporated and dissolved in water. Primers for reverse transcription, contained C6-aminolinker at 5′-end, were treated, in next step, with 80% acetic acid (3 h), precipitated in 1% sodium perchlorate solution in acetone and labelled with 5-FAM, 6-JOE, 5-ROX or 6-TAMRA (Anaspec). 300 µg of oligonucleotide was dissolved in water (11 µl) and 75 µl of 0.1M sodium tetraborate, pH 8.5 and fluorophore [TAMRA, FAM (200 µg) or JOE, ROX (250 µg)] in 14 µl DMSO was added. Reactions were incubated at 23 °C for 18 h by slowly mixing (150 rpm) then precipitated and run on 12% denaturing PAA gel. Oligonucleotides for ribonuclease H assay were purified using thin layer chromatography (TLC) and mobile phase was n-propanol/ammonia/water (52/35/13). Antisense oligonucleotides (2′-O-methyl RNA and 2′-O-methyl RNA containing LNA modifications (LNA - locked nucleic acids) were purified with TLC (short oligonucleotides) or denaturing 12% PAA gel (oligonucleotides longer than 11 nt). Oligonucleotides concentration was measured at 260 nm with Nanodrop spectrophotometer (Thermo Scientific). Molecular weights were confirmed by mass spectrometry (MALDI TOF, Autoflex, Brucker).