Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions

Abstract

Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.

New AdoMet Analogues as Tools for Enzymatic Transfer of Photo‐Cross‐Linkers and Capturing RNA–Protein Interactions

“…In ac omparativee valuation of all modified cap analogues used in this study,b inding testso fe IF4E with m 7 GTP and m 7 GpppA clearly showed the lowest K d values, as would be expected for the natural binding partners. The dissociation constant of m 7 GTP (approx.…”

“…The most widespread photo‐cross‐linking moieties available to date are aryl azides, benzophenones, and diazirines . Additionally, furan‐moieties allowed for the mild red‐light dependent nucleic acid inter‐strand and nucleic acid‐protein cross‐linking . DNA with diazirines at specific positions is accessible by solid‐phase synthesis and was used for inter‐strand photo‐cross‐linking as well as for cross‐linking to DNA‐binding proteins .…”

“…[5] Additionally,f uran-moieties allowed for the mild red-light dependent nucleic acid inter-strand [6] and nucleic acid-protein cross-linking. [7] DNA with diazirines at specific positions is accessible by solid-phase synthesis and was used for inter-strand photocross-linking [8] as wella sf or cross-linking to DNA-binding proteins. [9] DNA was also modified with four different photo-reactive moietiesi ncluding benzophenone andc ross-linked to yeast RNA polymerase III.…”

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes

“…Interstrand cross-link of DNA or RNA inhibits double strands from dissociating into single strands. 35 , 36 We previously developed a sequence-specific cross-link reaction that conjugates a pair of apurinic/apyrimidinic sites (AP sites) on complementary oligodeoxynucleotides (ODNs) using a short bifunctional cross-linker containing bis-aminooxy groups. 37 The cross-link formation makes the duplex rigid 38 and provides stable DNA scaffolds where enzymes are immobilized to catalyze glucose.…”

Synthesis and Improved Cross-Linking Properties of C5-Modified Furan Bearing PNAs

“…Unfortunately, these methodologies often require harsh reaction conditions [ 40 , 42 ], long reaction times [ 38 ], or impart problems like unspecific [ 37 , 42 ] and reversible [ 41 ] crosslinking, toxicity [ 39 ], and cellular damage [ 37 , 40 , 42 ]. In our research group, a furan oxidation-based crosslinking strategy was developed and successfully implemented for interstrand crosslinking of nucleic acids [ 43 , 44 ], crosslinking of DNA and RNA with proteins [ 45 ], and crosslinking of peptides with proteins [ 46 ] while ensuring a high selectivity and crosslinking yield. Furan oxidation can either be achieved via N -bromosuccinimide or via a biocompatible method using singlet oxygen.…”