Entwicklung von photo-reaktiven Sonden zum Einfangen von G-Quadruplex-DNA im Komplex mit endogenen Proteinen und exogenen Liganden
Zusammenfassung der Projektergebnisse
G‐quadruplexes (G4) are nucleic acid secondary structures that can form in guanine‐rich regions and are believed to be involved in key genome functions (replication, transcription, recombination and telomere maintenance). In this line, a small number of G4‐interacting proteins have been identified that bind, stabilize or resolve G4 structures. Nonetheless, our understanding concerning biological functions of G4, in particular on their interactions with proteins in a cellular environment remains limited. Initially we planned to identify new G4‐binding proteins using trifunctional capture compounds with G4 as bait, an aromatic azide for photo‐crosslinking and biotin for subsequent isolation of captured proteins with streptavidin‐coated magnetic beads. However, photo‐crosslinking yield with nucleolin, a well‐known G4‐binding protein, was very low. In addition, we observed that nucleolin binds very strongly to the capture compounds even without photo‐crosslinking allowing stringent washings. We, therefore, abandoned photo‐crosslinking and simply used bifunctional pull‐down probes with G4 as bait and biotin to isolate bound proteins. As biological relevant G4‐forming sequence we chose the G‐rich sequence Pu27 from the c‐myc promoter. Initial characterization of Pu27 oligodeoxynucleotide by size‐exclusion chromatography indicated that this sequence predominately forms multimeric structures. These structures are very stable and partly resist to commonly used thermal denaturation at 95 °C. Faced with this problem we developed an alternative chemical method by denaturation under strongly basic conditions and renaturation by neutralization. This method reliable yields monomeric structures and should be useful for other researchers working with G4 oligodeoxynucleotides. With these results in hand we turned to isolate and identify G4‐binding proteins from a human cell lysate by pull‐down and proteomic nano‐LC/MS analysis. Typically hundreds of proteins were identified and it was necessary to establish suitable control pull‐downs to select proteins which specifically bind to G4. Preferential binding to two different G4‐ODN compared to a non‐structured ODN (linear control), displacement by a G4‐binding small molecule ligand (competition control) and nuclear localization of target proteins were applied as selection criteria to obtain a list with 10 candidate proteins. One of these proteins, the ATP‐dependent RNA helicase DHX36 (RHAU), is a well characterized G4‐binding protein and we demonstrated that two more proteins from this list preferentially bind to G4‐DNA as well as G4‐RNA in vitro. Expression of more candidate proteins and biochemical/biophysical G4‐binding studies are on its way. Taken together the project has been successful, opens perspectives for future research and should lead to a better understanding of G4 biology.
Projektbezogene Publikationen (Auswahl)
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Non‐Canonical G‐quadruplexes Cause the hCEB1 Minisatellite Instability in Saccharomyces cerevisiae, eLife 2017, 6, e26884
A. Piazza, X. Cui, M. Adrian, F. Samazan, B. Heddi, A.‐T. Phan, A. G. Nicolas
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Probing of G‐Quadruplex Structures via Ligand‐Sensitized Photochemical Reactions in U‐Br‐Substituted DNA, Sci. Rep. 2018, 8, 15814
A. Saha, S. Bombard, A. Granzhan, M.‐P. Teulade‐Fichou
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Isolierung und Identifikation von G‐Quadruplex‐DNA‐bindenden Proteinen, Dissertation, RWTH Aachen University, 2019
V. Rauser
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Thermodynamically stable and genetically unstable G‐quadruplexes are depleted in genomes across species, Nucleic Acids Res. 2019, 47, 6098‐113
E. P. Lombardi, A. Holmes, D. Verga, M.‐P. Teulade‐Fichou, A. Nicolas, A. Londono‐Vallejo
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Nucleolin Discriminates Drastically between Long‐Loop and Short‐Loop Quadruplexes, Biochemistry 2020, 59, 1261‐72
A. Saha, P. Duchambon, V. Masson, D. Loew, S. Bombard, M.‐P. Teulade‐Fichou
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Quantitative Formation of Monomeric G‐Quadruplex DNA from Multimeric Structures of c‐Myc Promoter Sequence, ChemBioChem 2020
V. Rauser, E. Weinhold
