Wickramathilaka Malithi P, Tao Bernard Y
Department of Agricultural and Biological Engineering, Purdue University, 225 S. University Street, 745 Agricultural Mall Drive, West Lafayette, IN 47906 USA.
J Biol Eng. 2019 Jul 10;13:63. doi: 10.1186/s13036-019-0191-2. eCollection 2019.
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% ( = 10). This study demonstrates a convenient one pot strategy for crosslinking biological molecules.
开发了一种源自传统1-乙基-3-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)交联方法的改良策略，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，磷酰胺化ssDNA偶联物在数天内稳定，且磷酰胺化仅发生在ssDNA的5'磷酸基团上。开发了一种带有紫外检测的反相高效液相色谱(RP-HPLC)方法来测定偶联物的产率。该方法的变异系数(%CV)小于6%，偏差范围为-5.1 - 1.2%。通过传统EDC方法得到的偶联物产率为68.3 ± 2.2%，而改良的EDC/咪唑方法的产率为79.0 ± 2.4% (n = 10)。本研究展示了一种用于交联生物分子的便捷单步策略。
