Research paperTargeting human Rad51 by specific DNA aptamers induces inhibition of homologous recombination
Introduction
Homologous recombination (HR) is an evolutionarily conserved mechanism for the repair of double-strand breaks in DNA, and is also involved in DNA segregation and the creation of gene diversity [1], [2], [3], [4], [5], [6]. A key component of HR in eukaryotes is the protein Rad51, which catalyzes strand exchange between homologous DNA.
This protein acts against radio- and chemo-therapies by repairing the DNA damage caused by these treatments to kill cancer cells. It also participates in the proliferation and creation of more malignant tumors. Furthermore, Rad51 is frequently over-expressed in cancer cells and its amount correlates in some way with tumor resistance to chemotherapy and the advancement of cancer [7], [8], [9], [10]. Inhibition of the cellular production of Rad51 by anti-sense and siRNA or ribozyme strategies slows down tumor development and increases the efficiency of anti-cancer treatments [11], [12], [13]. These data have revealed Rad51 as an attractive target for the development of inhibitors for anti-cancer therapies.
Aptamers are nucleic acid molecules that bind to a specific molecular target. These ligands are derived from combinatorial libraries through an in vitro selection strategy called Systematic Evolution of ligands by EXponential enrichment (SELEX) [14], [15]. They are structured single-stranded RNA or DNA, which bind tightly to the target molecules according to their 3-D structure. Oligonucleotide analogs can be employed, for example 2′-fluoro-pyrimidines, 2′-amino-pyrimidines, 2′-O-methyl pyrimidines, 2′-hydroxy-purines, and phosphorothioate [16]. These modifications can significantly increase aptamer functional half-life and thus in vivo exposure, especially when they are destined to have a potential therapeutic use. Thus, aptamers are particularly interesting molecules, able to bind to a variety of targets including proteins, peptides, enzymes, cell surface receptors, etc. [16]. They are considered to be good candidates in the development of new therapeutic agents [17], [18], [19].
In the work reported here, we applied the SELEX approach to HsRad51 and identified DNA aptamers capable of binding HsRad51 and inhibiting its strand exchange activity. We also showed their ability to affect strongly the HsRad51 filament formation on DNA, which is the first step of the strand exchange reaction. Selected aptamers were also shown to be specific towards HsRad51, as they did not interact with the prokaryotic ortholog RecA at low concentrations. These results reveal new analytical tools for research to study HR and open up new perspectives for the development of novel therapies targeting Rad51.
Section snippets
Proteins and peptides
The HsRad51 and EcRecA proteins were prepared as described previously [20], [21]. The peptide of 28 amino-acids derived from the BRC4 motif of BRCA2 protein (BRC4-28) was obtained from NeoSystem (France) [20].
Oligonucleotides
The oligonucleotide library for aptamer selection and the following oligonucleotides for the strand exchange reaction were obtained from MWG (Germany) and used without further purification. Aptamers and their fragments used for the biochemical studies were also chemically synthesized by
Selection of DNA aptamers directed against HsRad51
The selection of HsRad51-binding oligonucleotides was carried out using purified HsRad51 as the target protein and starting from an oligonucleotide library. As ATP is present in cells and required for HsRad51 activity, it was routinely added. The library was composed of oligonucleotides that are 94 nucleotides in length. They contained two fixed sequences of 22 nucleotides, one on each side of a central random domain of 50 nucleotides. Selection of oligonucleotides bound to HsRad51 was
Conclusion
By using the SELEX approach to target entire HsRad51, we selected three aptamers that inhibit its DNA strand exchange activity. These aptamers were able to disassemble the HsRad51/DNA filament and inhibit HsRad51 activity more efficiently than other inhibitors, such as the BRC4 peptide [20]. Furthermore, aptamers were shown to be selective for HsRad51. Finally, various experiments suggested that aptamers may interact with HsRad51 by their particular 3-D structure.
These observations support the
Acknowledgements
We thank Dr. Christophe Thiriet and Dr. Jullien Drone for valuable discussions and careful reading of the manuscript. The authors acknowledge the IMPACT Core facilities – Biogenouest. This work was supported by grants from the Association pour la Recherche sur le Cancer [grant number 3862] and the Region de Pays de la Loire [grant names CIMATH and MIAPS]. S.F.M. was a recipient of a PhD fellowship from CONICYT (Chile) and the French Embassy.
References (29)
- et al.
Human Rad51 protein promotes ATP-dependent homologous pairing and strand transfer reactions in vitro
Cell
(1996) - et al.
Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein
Cell
(1992) - et al.
Homologous recombination and cell cycle checkpoints: Rad51 in tumour progression and therapy resistance
Toxicology
(2003) - et al.
Increased expression of human DNA repair genes, XRCC1, XRCC3 and RAD51, in radioresistant human KB carcinoma cell line N10
Oral Oncol.
(1998) - et al.
In vitro and in vivo potentiation of radiosensitivity of malignant gliomas by antisense inhibition of the RAD51 gene
Biochem. Biophys. Res. Commun.
(1998) - et al.
Aptamer therapeutics advance
Curr. Opin. Chem. Biol.
(2006) - et al.
Binding of recA protein to Z-form DNA studied with circular and linear dichroism spectroscopy
J. Biol. Chem.
(1989) - et al.
DNA aptamers that bind to chitin
Bioorg. Med. Chem. Lett.
(2000) - et al.
Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes
Proc. Natl. Acad. Sci. U S A
(1995) - et al.
Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
EMBO J.
(1998)
Catalysis of ATP-dependent homologous DNA pairing and strand exchange by yeast RAD51 protein
Science
Overexpression of Rad51 protein stimulates homologous recombination and increases resistance of mammalian cells to ionizing radiation
Nucleic Acids Res.
Chlorambucil induction of HsRad51 in B-Cell chronic lymphocytic leukemia
Clin. Cancer Res.
Over-expression of wild-type Rad51 correlates with histological grading of invasive ductal breast cancer
Int. J. Cancer
Cited by (10)
Dual and Opposite Effects of hRAD51 Chemical Modulation on HIV-1 Integration
2015, Chemistry and BiologyCitation Excerpt :Imatinib was purchased from Selleck Chemicals (Euromedex). Aptamers A30 and A47, previously selected as ligands of hRAD51 (Martinez et al., 2010), and their shortened versions A30c and A47c were purchased from MWG. Cisplatin was purchased from Sigma.
Targeting homologous recombination, new pre-clinical and clinical therapeutic combinations inhibiting RAD51
2015, Cancer Treatment ReviewsCitation Excerpt :RAD51 is able to bind both DNA and RNA therefore it can be effectively inhibited using aptamers. DNA aptamers are comprised of oligonucleotides capable of entering tissues and binding a specific molecular target [55]. Using the Systematic Evolution of Ligands by Exponential enrichment (SELEX) approach followed by a strand exchange assay Martinez and colleagues identified three DNA aptamers (A13, A30 and A79) capable of interacting specifically with RAD51 with IC50 values in the clinically relevant range of 20–25 nM [55].
Identification and characterization of human Rad51 inhibitors by screening of an existing drug library
2014, Biochemical PharmacologyCitation Excerpt :Thus, the overarching goal of our study was to develop new inhibitor of Rad51 to overcome resistance of anticancer therapy. We previously identified peptide [27,34] or oligonucleotide [35] inhibitors of Rad51 and we chose now in this study to identify small chemical molecules. Therefore, here, 1120 molecules, provided by Prestwick chemical, were tested for their inhibitory effect on the strand exchange reaction catalyzed by Rad51.
Targeting RAD51 phosphotyrosine-315 to prevent unfaithful recombination repair in BCR-ABL1 leukemia
2011, BloodCitation Excerpt :Moreover, TKIs may not significantly affect BCR-ABL1 kinase and its signaling pathways in LSCs and also in LPCs in the presence of physiologic concentrations of growth factors or in a protective environment, such as bone marrow niche.5,45 Because up-regulation of RAD51 recombinase may cause genomic instability, several approaches have been developed to inhibit/control its activity, for example, protein aptamer derived from BRC4 motif of BRCA2 to inhibit RAD51 filament formation and DNA aptamer and a chemical compound that inhibit RAD51-dependent pairing and strand exchange.46-48 However, these strategies will also target RAD51-mediated HomoRR in normal cells, which may exert a deleterious effect on cell survival and chromosomal stability.49
Synthetic Lethality through the Lens of Medicinal Chemistry
2020, Journal of Medicinal ChemistryMetformin overcomes resistance to cisplatin in triple-negative breast cancer (TNBC) cells by targeting RAD51
2019, Breast Cancer Research