期刊
JOURNAL OF BIOLOGICAL ENGINEERING
卷 13, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s13036-019-0191-2
关键词
EDC crosslinking; MALDI-TOF; RP-HPLC; Product yield; Reaction mechanism
资金
- Indiana Soybean Alliance
- Purdue University Department of Agricultural and Biological Engineering
- USDA National Institute of Food and Agriculture (Hatch) project [1002722]
- College of Engineering Graduate Research Ross Fellowship
- NIFA [690292, 1002722] Funding Source: Federal RePORTER
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 aconvenientone pot strategy for crosslinking biological molecules.
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