Atto680 nhs ester

Herceptin^® (trastuzumab), a Food and Drug Administration-approved humanized anti-HER2 IgG₁ monoclonal antibody, was obtained from Roche Pharma (Basel, Switzerland). Amine-reactive ATTO655-N-hydroxysuccinimidyl ester (ATTO655-NHS ester; λ_ex/λ_em: 663/684), ATTO680-NHS ester (λ_ex/λ_em: 680/700), ATTO700-NHS ester (λ_ex/λ_em: 700/719), and free ATTO680-COOH were purchased from ATTO TEC (Siegen, Germany). NHS-Fluorescein (λ_ex/λ_em: 494/518), Alexa Fluor^® 488-NHS ester (λ_ex/λ_em: 494/518), and Alexa Fluor^® 680-NHS ester (λ_ex/λ_em: 679/702) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). L-Trptophan and L-methionine were purchased from Sigma-Aldrich (St. Louis, MO, USA). N-Acetyl-L-Tyrosine and L-histidine were purchased from Tokyo Chemical Industry (Tokyo, Japan).

5 nmol of amino-modified RNA was dissolved in 50 μl of a 50 mM HCO₃^– buffer (pH 8.2). 25 μg of the ATTO680-NHS ester (ATTO-TEC GmbH) was dissolved in an equal volume of DMF and was added to the RNA. The mixture was incubated for 1 h at room temperature, before precipitation of the RNA with 70% ethanol and purification via reversed phase HPLC (ÄKTA purifier; column: 250/4 nucleodur C18; gradient: 5% to 30% acetonitrile in 0.1 M triethylammonium acetate buffer (pH 7.5) over 60 min; flow rate: 0,5 ml/min).