Pyrrolidinyl peptide nucleic acid probe capable of crosslinking with DNA

Abstract

DNA interstrand cross-linking (ICL) is a phenomenon in which two strands of the DNA (most often as part of the same DNA duplex) bind together via a covalent bond. This process prevents the DNA strand dissociation, which is essential for its biological functions, and thus is extremely damaging to cells. To overcome the problem of non-specificity associated with general DNA cross-linking agents, a cross-linkable probe in which the reactive group is placed on the probe that can specifically recognize part of the DNA target. The strong binding affinity and biological stability of the pyrrolidinyl peptide nucleic acid (acpcPNA) inspired us to develop a new activate-on-demand cross-linkable acpcPNA probe with furan as the reactive moiety, which can be activated by an oxidative process. A simple post-synthetic modification protocol was developed to synthesize such probes and their cross-linking with DNA were studied by thermal denaturation, denaturing PAGE, reverse phase HPLC, and MALDI-TOF MS. The work was divided into two sections. In section I, the effect of the furan position on the acpcPNA strand towards the cross-linking efficiency was investigated. The probe was designed so that the furan moiety was attached to the probe independent from the nucleobase so that it does not interfere with the normal base-pairing process. The results revealed that terminally furan-modified acpcPNA could undergo cross-linking with C > A >> G while the internally furan-modified acpcPNA could not. The G-base was found to participate in such cross-linking with furan for the first time. The C-inserted DNA sequences could enhance the cross-linking efficiency of the internally furan-modified probe. It was concluded that the essential factor of the successful cross-linking reaction is the availability of the nucleobase nearby the furan activated moiety in addition to the ability of the probe to form a stable hybrid with the target. In section II, the cross-linking efficiency of the acpcPNA and aegPNA modified with various furan building blocks at the terminal were compared. The results showed that various furan building blocks and various PNA types of probe offered different cross-linking efficiency and selectivity. In most cases, aegPNA probes showed somewhat higher cross-linking yields over acpcPNA probes, but the latter showed a higher selectivity toward C-base. In addition, a study of internally cross-linking with acpcPNA probes was performed with various DNA targets, and the cross-linking reaction was most efficient when there was a free C-base present at or adjacent to the opposite position of the furan building block. Overall, the major conclusion obtained from the study is that both the ability to form a stable duplex and the availability of the unpaired DNA nucleobase with an exocyclic amino group (A, C, G) were essential for the cross-linking reaction, which provides further insights into the requirements for an efficient ICL formation.