Furan‐Oxidation‐Triggered Inducible DNA Cross‐Linking: Acyclic Versus Cyclic Furan‐Containing Building Blocks—On the Benefit of Restoring the Cyclic Sugar Backbone

Abstract

Oligodeoxynucleotides incorporating a reactive functionality can cause irreversible cross-linking to the target sequence and have been widely studied for their potential in inhibition of gene expression or development of diagnostic probes for gene analysis. Reactive oligonucleotides further show potential in a supramolecular context for the construction of nanometer-sized DNA-based objects. Inspired by the cytochrome P450 catalyzed transformation of furan into a reactive enal species, we recently introduced a furan-oxidation-based methodology for cross-linking of nucleic acids. Previous experiments using a simple acyclic building block equipped with a furan moiety for incorporation into oligodeoxynucleotides have shown that cross-linking occurs in a very fast and efficient way and that substantial amounts of stable, site-selectively cross-linked species can be isolated. Given the destabilization of duplexes observed upon introduction of the initially designed furan-modified building block into DNA duplexes, we explore here the potential benefits of two new building blocks featuring an extended aromatic system and a restored cyclic backbone. Thorough experimental analysis of cross-linking reactions in a series of contexts, combined with theoretical calculations, permit structural characterization of the formed species and allow assessment of the origin of the enhanced cross-link selectivity. Our experiments clearly show that the modular nature of the furan-modified building blocks used in the current cross-linking strategy allow for fine tuning of both yield and selectivity of the interstrand cross-linking reaction.

Probing DNA interstrand cross-link formation by an oxidized abasic site using nonnative nucleotides

“…Similarly, dG does not form ICLs with dioxobutane, or the bis-electrophile derived from furan oxidation, which are also 1,4-dicarbonyl molecules. 5,33,34 …”

Reversibly locked thionucleobase pairs in DNA to study base flipping enzymes

“…While the cross-linking reaction itself is straight-forward, the vinyl-substituted nucleosides need to be chemically synthesized and subsequently converted into their phosphoamidites for chemical DNA synthesis. The same holds true for cross-links based on aldehyde [ 8 , 35 – 37 ] or click chemistry [ 38 ]. In addition, long linkers that might interfere with protein binding are typically employed.…”

Photouncaged Sequence-specific Interstrand DNA Cross-Linking with Photolabile 4-oxo-enal-modified Oligonucleotides

“…The 4-oxo-enal moieties generated through oxidation of furan ring have been previously reported 13 21 22 23 for ICL formation between the exocyclic amine of adenine or cytosine and the aldehyde group of 4-oxo-enal moiety 13 21 22 23 . Different from previous 4-oxo-enal moieties that contain two carbonyl groups, we developed a 4-oxo-enal moiety with one carbonyl and one amide groups.…”

“…More promising results are achieved when a reactive cross-linking moiety is produced from a stable precursor, e.g. disulfide bonds 3 , quinone methides 6 11 14 , carbazoles 5 15 16 , benzophenone derivatives 17 , phenylselenyl derivatives of pyrimidines 18 , abasic site 19 20 , and furan derivatives 13 21 22 23 . Among them, Madder reported that methylene blue-induced 1 O 2 formation triggered furan oxidation for further DNA cross-linking 13 21 22 .…”

“…disulfide bonds 3 , quinone methides 6 11 14 , carbazoles 5 15 16 , benzophenone derivatives 17 , phenylselenyl derivatives of pyrimidines 18 , abasic site 19 20 , and furan derivatives 13 21 22 23 . Among them, Madder reported that methylene blue-induced 1 O 2 formation triggered furan oxidation for further DNA cross-linking 13 21 22 . And Sasaki et al described an alternative cross-linking strategy through oxidative deprotection of reactive vinyl groups 24 25 26 27 28 29 30 .…”