Abstract
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.
Highlights
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
Phosphoramidated single stranded DNA (ssDNA) conjugate analysis; MALDI-TOF Conjugates formed via the conventional EDC, and the EDC and imidazole (EDC/Im) reaction mechanisms were characterized by MALDI-TOF
A novel phosphoramidated ssDNA was synthesized as a proof-of-concept modality to compare the conventional carbodiimide (EDC) reaction with an adapted strategy using EDC and imidazole (EDC/Im)
Summary
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). 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide (EDC) is a water soluble zero-length crosslinker which is convenient to use and is relatively inexpensive It has been used in a variety of conjugation techniques to couple carboxyl groups to primary amines [1,2,3]. The O-acylisourea product intermediate can be hydrolyzed, reverting to the original carboxylate molecule [1] To overcome this limitation, sulfo NHydroxysuccinimide (sulfo-NHS ester) has been used to form a more stable second intermediate prior to amination [5]. This two-step process faces the risk of hydrolyzing the NHS intermediate, as it has a half-life ranging from 10 min – 1 h [16] Is this two-step process cumbersome, it can be prone to poor product yields during reactions with enzymes or immunoglobins due to loss of activity.
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