A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA

Abstract

Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3′-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3′-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

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

“…Furthermore, N 4 modification could complement the already known photocaging strategy and become a great tool for the regulation of gene expression. Considering the crosslinking or photo‐immobilisation possibilities of AcBz or BzBz functional group containing DNA demonstrated earlier, the demodification step would enable the separation of modified DNA and the covalently linked substrate to delve more deeply into the bioconjugation mechanism. Moreover, given that, in some cases, the modified ONs can be incompatible with downstream amplification procedures (e.g., PCR or reverse transcription), there might be a necessity to remove modifications, while leaving the whole nucleic acid molecule unaffected.…”

Photochemical modifications for DNA/RNA oligonucleotides

“…84 For example, terminal deoxynucleotidyl transferase enzyme could be crosslinked to 3 0 -tails of DNA containing benzophenones and acetophenones on N 4 -dC's using 365 nm light (Fig. 8) 87 Reaction characteristics. UV irradiation (365 nm) of benzophenones generates C-O biradical through n-p* transition, which can lead either to photocrosslinking or its reversal, photocleavage.…”

“…86 On the other hand, benzophenone-and acetophenone-modications on N 4 -dC could generate protein-DNA crosslinks with the bound terminal deoxynucleotidyl transferases using 365 nm light. 87…”

Recent Advances in Chemical Biology Using Benzophenones and Diazirines as Radical Precursors

“…This product was proposed based on NMR and UHPLC-MS/MS analysis of the byproducts following irradiation. In a dual crosslinking and immobilization approach, Jakubovska et al [72] functionalized N 4 -cytidines with BP and demonstrated the dual usefulness of the installed BP. Through BP-mediated crosslinking, they successfully (1) immobilized the modified oligonucleotides to polymeric solid supports and (2) covalently labelled interacting proteins of the oligonucleotide.…”