Enzymatic PEGylation of oligonucleotides

The gene and RNAi therapies as well as therapeutic aptamers attracted a great interest owing to the potential high selectivity and the theoretical possibility to treat a wide range of diseases, even those that have no other therapies. Unfortunately, this promise was partially reconsidered in face of the many difficulties related to the in vivo application of these molecules and the issues associated with their stability and delivery. Unmodified DNAs are rapidly degraded by 3’ exonucleases [1] and, furthermore, they suffer a fast kidney clearance and scarce cell internalization. To address these limitations, two main approaches of nucleic acids delivery have been proposed i.e. viral vectors and lipidic or polymeric non-viral vectors. PEGylation, the covalent attachment of polyethylene glycol (PEG) [2], has been proposed for prolonging the pharmacokinetic profile of nucleic acid materials: PEGylated oligonucleotides and siRNAs have demonstrated an improved cell internalization and stability with respect to the free nucleic acids [3, 4]. A method for the direct selective conjugation of PEG to nucleic acid would be desired to overcome the constrains of the nucleic acids PEGylation based on the covalent attachment of a properly activated PEG to modified DNA or RNA oligonucleotides. In the field of protein PEGylation, the attention has been recently directed towards the development of enzymatic methods of conjugation [2, 5]. These new perspectives in protein PEGylation inspired us to develop a method consisting of an enzymatic PEGylation of oligonucleotides. T4 DNA ligase enzyme was used to ligate double stranded oligonucleotides to a 20kDa PEG bearing a short oligonucleotide sequence. The chemical synthesis of the oligo-PEG conjugate was characterized and standardized. Hence, the enzymatic ligation of the oligo-PEG conjugate to a dsDNA was performed using a set of appositely designed oligonucleotides as model system. The ligation protocol was optimized allowing the complete achievement of a stable PEGylated dsDNA system. This early study on enzymatic PEGylation of oligonucleotides can potentially be applied to every DNA or RNA sequence and can stimulate new researches in this field.