Photo-Cross-Linked Oligonucleotide Duplex as a Decoy-DNA for Inhibition of Restriction Endonuclease Activity

As a novel type of regulator molecule for DNA-recognizing proteins, a photo-cross-linked oligonucleotide duplex was designed and synthesized. The molecule regulated the activity of a restriction endonuclease by being recognized as a substrate. This type of regulating molecule is regarded as a decoy-DNA. 4,5′,8-[4-Aminoethylaminomethyl]-trioxalen (aeAMT) was conjugated with an oligodeoxyribonucleotide (ODN) at the 5′-end and the aeAMT was cross-linked with the thymine residue of the complementary oligonucleotide upon UVA irradiation. The terminally cross-linked oligonucleotides, singly clipped (SC) decoy-DNA, acquired thermal stability. An oligonucleoside phosphorothioate (OPT) was also introduced as one or both components, yielding three types of decoy-DNAs, SC-ODN-ODN (SC.DD), SC-OPT-ODN (SC.SD), and SC-OPT-OPT (SC.SS). The SC decoy-DNAs inhibited the function of the restriction endonuclease, AatII, in a sequence-specific and concentration-dependent manner with an appreciable IC50 value (1.3 μM for SC.DD, 0.016 μM for SC.SD, 0.002 μM for SC.SS). The SC decoy-DNAs were found to be effective for regulating the DNA recognizing proteins.