S acetylthioglycolic acid nhs ester a9043

For 5’ modification and PS bond modification: primers with 2PS bonds, Acrydite™, C6-Alex 647, amine C6 and amine C12 were ordered from IDT. Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588). Biotin NHS ester (H1759), azidoacetic acid NHS ester (900919), s-acetylthioglycolic acid NHS ester (A9043), and propargyl NHS ester (764221) were purchased from Sigma-Aldrich. NHS esters were incubated with 10 μM primers with 5’ amine C6 or C12 group at 1 mM concentration in 1 x borate buffer (Thermo Fisher, 28341) overnight at room temperature. Primers were then desalted using Bio-Spin 30 columns (Bio-rad). The labelling efficiencies were measured by HPLC and MALDI-TOF mass spectrometry (Supplementary Fig. 3). For 3’ Am-ddU modification: An aliquot of 10 μg dsDNA donors, 20 μM amino-11-ddUTP (Lumiprobe), 50 μM CoCl₂, and 1 U TdT polymerase (New England Biolabs), 1 x TdT reaction buffer were incubated in a 50 μl reaction at 37 °C for 4 h. amino-11-ddUTP modified dsDNA was purified using QlAquick PCR purification kit (Qiagen) after stopping the reaction with 10 μl 0.2 M EDTA (PH = 8.0). The resulting product was then used to generate 3’ ddU-PEG10 modified dsDNA by incubation with Bis-PEG10-NHS ester at conditions described above.