Oligonucleotides using carbodiimides

An improved system for the delivery of antisense oligonucleotides uses a carbodiimide mediated covalent conjugate with a copolymer of vinyl pyrrolidone and 2-hydroxyethyl methacrylate. ... 

Figure 1.72 Cystamine may be used to label phosphate groups, such as at the 5 -end of oligonucleotides, via a carbodiimide reaction using EDC. The resultant phosphoramidate linkage is a common way to modify oligonucleotides at the 5 -end.

Molecules containing phosphate groups, such as the 5 phosphate of oligonucleotides, also may be conjugated to amine-containing molecules by using a carbodiimide-mediated reaction (Chapter 27, Section 2.1). The carbodiimide activates the phosphate to an intermediate phosphate ester similar to its reaction with carboxylates (Chapter 3, Section 1). In the presence of an amine, the ester reacts to form a stable phosphoramidate bond (Reaction 13). 

The following protocol assumes the prior derivatization of an oligonucleotide at the 5 end using a bis-hydrazide compound according to the protocol of Section 2.1 (this chapter) using a carbodiimide-mediated reaction. 

Phosphate-containing carbohydrates that are stable, such as the 5 -phosphate of the ribose derivatives of oligonucleotides, may be targeted for modification using a carbodiimide-facilitated reaction (Section 4.3). The water-soluble carbodiimide EDC can react with the phosphate groups to form highly reactive phospho-ester intermediates. These intermediates can react with amine- or hydrazide-containing molecules to form stable phosphoramidate bonds. 

This procedure was introduced by Halloran and Parker for use with mono- and oligonucleotides. Humayun and Jacob modified it to reduce the amount of insoluble aggregate they obtained with the original method, especially with purine nucleotides. In a reaction following the modified procedure, about 0.25 mmol of solid l-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide is added to 0.1 mmol of nucleotide in 0.5 ml of water. The pH is adjusted to 7 with dilute NaOH if necessary, and the solution is incubated at 60° for 10 min. The mixture is then added dropwise to a protein solution of 10-20 mg in 0.2-0.4 ml of 0.15 M NaCl, and this solution is kept in the dark overnight at room temperature. The product is then separated from the free nucleotide and the hydrolyzed carbodiimide reagent by dialysis or gel filtration. 

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