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

Abstract

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.