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

Abstract

In 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 hydrogenbonded 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 Cinsertion) was present in the complementary DNA strand close to the furan modification site.

Synthesis and structure-activity relationship of Peptide Nucleic Acid Probes with improved Interstrand-Crosslinking abilities: Application to Biotin-mediated RNA-Pulldown

“…PNA monomer and probe design In previous work, we reported that the crosslinking ability of furan-containing PNA probes towards target oligonucleotides was negatively affected by nucleobase pairing. 23,28,29 To understand whether that behaviour was connected to the reduced nucleophilicity of the target nucleophile or to an unfortunate placement of the pro-reactive units on the PNA strand, we decided to explore the possibility to improve the ICL yield by expanding the toolbox of furan-containing monomers. Next to furan introduction through the universal nucleobase building block M1, we explored the possibility to append the small aromatic ring on the backbone of a PNA building block, as well as mounted on a modified nucleobase, designing the monomers M2 and M3 respectively.…”

“…next position toward the N-terminus, Figure 2b). Next, in analogy to what was recently reported by Vilaivan's group, 29 we additionally evaluated the effect of furan-introduction by incorporating M1 or M2 as a terminal modification, next to introduction of a simple furan unit, introduced via the building block M4 (Figure 2c). PNA monomer synthesis Temporary protection of the furan ring to avoid alkylation of the C5 position during PNA cleavage from the solid support was earlier demonstrated to minimize the formation of byproduct, thus increasing the final yield of synthesis.…”

A Photosensitized Singlet Oxygen (1O2) Toolbox for Bio-Organic Applications: Tailoring 1O2 Generation for DNA and Protein Labelling, Targeting and Biosensing

“…Beside the original purpose of ICL and DNA/RNA alkylation, furan technology was also exploited for the covalent immobilization of oligonucleotides on surface, as demonstrated by SPR measurements [ 177 ]. This chemistry was at the same time extended towards unnatural oligonucleotide derivatives, such as PNAs, for the realization of PNA-DNA ( Figure 12 A) CL systems, using NBS to trigger the furan oxidation, and further adopted by the Vilaivan group for the realization of acpc (pyrrolidinyl)-PNA probes able to cross-link to a DNA target [ 178 ]. Finally, we exploited furan chemistry for the realization of an oligonucleotide-templated PNA–PNA ligation reaction, which can be conveniently used for the realization of constructs on surface as well as for the detection of short oligonucleotides [ 179 ].…”

Synthesis and structure–activity relationship of peptide nucleic acid probes with improved interstrand-crosslinking abilities: application to biotin-mediated RNA-pulldown

“…In previous work, we reported that the crosslinking ability of furan-containing PNA probes towards target oligonucleotides was negatively affected by nucleobase pairing. 22,27,28 To understand Fig. 1 Schematic representation of the experiment proposed in this work.…”

“…2b). Next, in analogy to what was recently reported by Vilaivan's group, 28 we additionally evaluated the effect of furan-introduction by incorporating M1 or M2 as a terminal modification, next to introduction of a simple furan unit, introduced via the building block M4 (Fig. 2c).…”