Ellen Gyssels

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

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 todays antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation methods 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cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes.

Photoinduced Cross-Linking of Short Furan-Modified DNA on Surfaces

We report for the first time the formation of site-specific interstrand cross-linked (ICL) surface-immobilized furan-modified DNA duplexes via singlet oxygen. 1O2, necessary for effecting furan-mediated ICL formation, was produced in situ using methylene blue or a zinc phthalocyanine derivative (TT1) as a photosensitizer. Via surface plasmon resonance spectroscopy, we show that surface ICL was achieved, and a robust link formed that enhances the stability of the 12-mer duplex even after surface regeneration. The described method represents a novel platform technology based on surfaces with addressable and stable DNA duplexes requiring only short oligonucleotides.

Triplex Crosslinking through Furan Oxidation Requires Perturbation of the Structured Triple-Helix

Short oligonucleotides can selectively recognize duplexes by binding in the major groove thereby forming triplexes. Based on the success of our recently developed strategy for furan‐based crosslinking in DNA duplexes, we here investigated for the first time the use of the furan‐oxidation crosslink methodology for the covalent locking of triplex structures by an interstrand crosslink. It was shown that in a triplex context, although crosslinking yields are surprisingly low (to nonexistent) when targeting fully complementary duplexes, selective crosslinking can be achieved towards mismatched duplex sites at the interface of triplex to duplex structures. We show the promising potential of furan‐containing probes for the selective detection of single‐stranded regions within nucleic acids containing a variety of structural motifs.

Hyphenation of a Deoxyribonuclease I immobilized enzyme reactor with liquid chromatography for the online stability evaluation of oligonucleotides

The stability of antisense oligonucleotides (ONs) toward nucleases is a key aspect for their possible implementation as therapeutic agents. Typically, ON stability studies are performed off-line, where the ONs are incubated with nucleases in solution, followed by their analysis. The problematics of off-line processing render the detailed comparison of relative ON stability quite challenging. Therefore, the development of an online platform based on an immobilized enzyme reactor (IMER) coupled to liquid chromatography (LC) was developed as an alternative for improved ON stability testing. More in detail, Deoxyribonuclease I (DNase I) was immobilized on epoxy-silica particles of different pore sizes and packed into a column for the construction of an IMER. Subsequently, the hyphenation of the IMER with ion-pair chromatography (IPC) and ion-exchange chromatography (IEC) was evaluated, leading to the successful development of two online methodologies: IMER-IPC and IMER-IEC. More specifically, natural and modified DNA and RNA oligonucleotides were used for testing the performance of the methodologies. Both methodologies proved to be simple, automatable, fast and highly reproducible for the quantitative and qualitative evaluation of ON degradation. In addition, the extended IMER life time in combination with a more straightforward control of the reaction kinetics substantiate the applicability of the IMER-LC platform for ON stability tests and its implementation in routine and research laboratories.

DNA Interstrand Cross‐Link Formation Using Furan as a Masked Reactive Aldehyde

This unit describes a method for interstrand cross‐linking between a furan‐modified oligonucleotide and its unmodified complement. The synthesis of two furan‐modified phosphoramidites, selected based on high cross‐linking yield versus improved cross‐linking selectivity, is described. The methods allow gram‐scale synthesis starting from stable and readily available furan derivatives. Cross‐linking requires selective oxidation of the furan moiety to an aldehyde. The masked nature of the latter avoids undesired and off‐target reactions, resulting in clean and high‐yield cross‐link formation. Curr. Protoc. Nucleic Acid Chem. 54:5.12.1‐5.12.16.

Interstrand Cross-Linking of Nucleic Acids: From History to Recent and Future Applications

This chapter contains an elaborate overview of various methods that have been developed for interstrand cross-linking of nucleic acids. The existing cross-link methodologies can be subcategorized in different groups, each interesting depending on the requirements of the envisaged application. An endogenous or exogenous bifunctional compound can react with two different nucleophilic groups on the nucleobases, resulting in fast and high-yielding cross-linking. However, when site selectivity of the formed cross-link is desired, other approaches are required. Therefore, a series of methodologies are at hand where functionalities are introduced in oligonucleotide probes, which can be intrinsically reactive or can be triggered and activated at a selected time upon target recognition. In a third class, both strands are modified with orthogonal groups. After reaction of the introduced functionalities, a cross-link is formed at a specific and fixed position. The chapter ends with an overview of and outlook to present and future applications. It is thus shown that cross-linking agents can be exploited as useful therapeutic or diagnostic tools and have further proven their utility in the stabilization or structure elucidation of cross-linked systems aimed at studying and understanding the repair systems in a cell.