Transient N⁴ -Acyl-DNA Protection against Cleavage by Restriction Endonucleases

A set of five N⁴ -acyl-modified 2'-deoxycytidine 5'-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N⁴ -acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N⁴ -acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N⁴ modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N⁴ -modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.