Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency

AbstractIn this study, we describe a furan‐modified acpcPNA as a probe that can form an interstrand crosslink (ICL) with its DNA target upon activation with N‐bromosuccinimide (NBS). To overcome the problem of furan instability under acidic conditions, a simple and versatile post‐synthetic methodology for the attachment of the furan group to the PNA probe was developed. Unlike in other designs, the furan was placed at the end of the PNA molecule or tethered to the PNA backbone with all the base pairs in the PNA ⋅ DNA duplexes fully preserved. Hence, the true reactivity of each nucleobase towards the crosslinking could be compared. We show that all DNA bases except T could participate in the crosslinking reaction when the furan was placed at the end of the PNA strand. The crosslinking process was sensitive to mispairing, and lower crosslinking efficiency was observed in the presence of a base‐mismatch in the PNA ⋅ DNA duplex. In contrast, when the furan was placed at internal positions of the acpcPNA ⋅ DNA duplex, no ICL was observed; this was explained by the inability of a hydrogen‐bonded nucleobase to participate in the crosslinking reaction. The crosslinking efficiency was considerably improved, despite lower duplex stability, when an unpaired base (in the form of C‐insertion) was present in the complementary DNA strand close to the furan modification site.