Characterization of covalent crosslinking strategies for synthesizing DNA-based bioconjugates.

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.