An adapted strategy from the conventional 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) crosslinking method was developed to form a covalently coupled phosphoramidated single stranded DNA (ssDNA). Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) results demonstrated that the phosphoramidated ssDNA conjugate is stable for several days, and that phosphoramidation occurred exclusively at the 5' phosphate of ssDNA. A reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection was developed to determine the yield of conjugates. The methods coefficients of variation (%CV) were less than 6%, and biases ranged from - 5.1 - 1.2%. The conjugate yield via the conventional EDC method was 68.3 ± 2.2%, while that of the adapted EDC/Imidazole method was 79.0 ± 2.4% (n = 10). This study demonstrates a convenient one pot strategy for crosslinking biological molecules.

中文翻译：

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用于合成基于DNA的生物共轭物的共价交联策略的表征。

从常规的1-乙基-3-（3-二甲基氨基丙基）碳二亚胺盐酸盐（EDC）交联方法开发了一种适应策略，以形成共价偶联的磷酰胺化单链DNA（ssDNA）。基质辅助激光解吸电离飞行时间（MALDI-TOF）结果表明，磷酸化的ssDNA共轭物稳定了几天，而磷酰胺化仅发生在ssDNA的5'磷酸上。建立了具有紫外检测功能的反相高效液相色谱（RP-HPLC）方法，以测定结合物的收率。方法的变异系数（％CV）小于6％，偏差范围为-5.1-1.2％。通过常规EDC方法的偶联物收率为68.3±2.2％，而经过改进的EDC /咪唑方法的偶联物收率为79.0±2.4％（n = 10）。