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(586a) Characterization and Evaluation of Zero-Length Covalent Crosslinking Strategies for DNA Based Applications

Authors: 
An adapted strategy for covalently coupling amine and phosphate functional groups in biological molecules was developed from the conventional 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) crosslinking method. The coupling mechanism was studied with synthetic single stranded DNA (ssDNA) and ethylenediamine (EDA). The study demonstrates that the phosphoramidated ssDNA conjugate is stable for up to 14 days upon storage at -20 °C. It also provides evidence for stable covalent phosphoramidation exclusively at the 5’ phosphate group of the ssDNA. Fourier transform infrared spectroscopy (FTIR) was utilized for bond characterization of the conjugates. The extent of reactions were determined by reversed phase liquid chromatographic methods (RP-HPLC). A comprehensive study on analytical method validation was conducted to determine parameters such as the accuracy and detection limits of the developed HPLC analytical method. Finally, we report the yield of phosphoramidated products synthesized through the two separate crosslinking strategies, and provide a conclusive comparison between the two reaction approaches.

The current study on crosslinking strategies using EDC and imidazole is presented as a convenient one-pot reaction method for areas of research where there is a great lack of flexibility in altering reaction conditions such as the temperature or pH, to obtain higher product yields. It is directly applicable to studies in biomedical diagnostics, biosensor development, and self-assembled biomaterial synthesis.

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