Tetramethylrhodamine 6 maleimide

The 3’-amino and 5’- C6SSC6 disulfide modified oligonucleotide (Axolabs, Kulmbach, Germany) was dissolved in 100 mM sodium borate buffer containing 20% acetonitrile (pH 8.5) to a final concentration of 800 μM. Atto488-NHS ester (Atto-Tec, Siegen, Germany) was dissolved in anhydrous DMSO to a working concentration of 1 mM. Three molar equivalents of Atto488-NHS ester solution were added over 2 h every 15 min, following 3 h incubation at 25°C. The resulting construct was purified by EtOH precipitation and redissolved in water to a concentration of 1 mM. The C6SSC6 disulfide modified end was reduced with buffered tris(2-carboxyethyl)phosphine (TCEP, 700 times molar excess, Sigma Aldrich, Steinheim, Germany) for 2.5 h at RT. TCEP was removed by EtOH precipitation. The remaining pellet was redissolved in 50 mM sodium phosphate buffer 20% acetonitrile (pH 7) to a concentration of 800 μM. Tetramethylrhodamine-6-maleimide (Life Technologies, Darmstadt, Germany) was dissolved in anhydrous DMSO to a working concentration of 1 mM. The Tetramethylrhodamine-6-maleimide solution (1.3 equivalents) was added immediately to the oligonucleotide solution, following incubation of 2 h at 25°C. The product was purified by EtOH precipitation and high-performance liquid chromatography.