Several sequences forming G-quadruplex are highly conserved in regulatory regions of genomes of different organisms and affect various biological processes like gene expression. Diverse G-quadruplex properties can be modulated via their interaction with small polyaromatic molecules such as pyrene. To investigate how pyrene interacts with G-rich DNAs, we incorporated deoxyuridine nucleotide(s) with a covalently attached pyrene moiety (Upy) into a model system that forms parallel G-quadruplex structures. We individually substituted terminal positions and positions in the pentaloop of the c-kit2 sequence originating from the KIT proto-oncogene with Upy and performed a detailed NMR structural study accompanied with molecular dynamic simulations. Our results showed that incorporation into the pentaloop leads to structural polymorphism and in some cases also thermal destabilization. In contrast, terminal positions were found to cause a substantial thermodynamic stabilization while preserving topology of the parent c-kit2 G-quadruplex. Thermodynamic stabilization results from π-π stacking between the polyaromatic core of the pyrene moiety and guanine nucleotides of outer G-quartets. Thanks to the prevalent overall conformation, our structures mimic the G-quadruplex found in human KIT proto-oncogene and could potentially have antiproliferative effects on cancer cells.

CEITEC Central European Institute of Technology Masaryk University Kamenice 5 CZ 62500 Brno Czechia

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 CZ 62500 Brno Czechia

EN FIST Centre of Excellence Trg OF 13 SI 1000 Ljubljana Slovenia

Faculty of Chemistry and Chemical Technology University of Ljubljana Večna pot 113 SI 1000 Ljubljana Slovenia

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 CZ 625 00 Brno Czechia

Slovenian NMR Centre National Institute of Chemistry Hajdrihova 19 SI 1000 Ljubljana Slovenia

See more in PubMed

Chambers V.S., Marsico G., Boutell J.M., Di Antonio M., Smith G.P., Balasubramanian S.. High-throughput sequencing of DNA G-quadruplex structures in the human genome. Nat. Biotechnol. 2015; 33:877–881. PubMed

Maizels N., Gray L.T.. The G4 genome. PLoS Genet. 2013; 9:e1003468. PubMed PMC

De Cian A., Lacroix L., Douarre C., Temime-Smaali N., Trentesaux C., Riou J.F., Mergny J.L.. Targeting telomeres and telomerase. Biochimie. 2008; 90:131–155. PubMed

Balasubramanian S., Neidle S.. G-quadruplex nucleic acids as therapeutic targets. Curr. Opin. Chem. Biol. 2009; 13:345–353. PubMed PMC

Edling C.E., Hallberg B.. c-Kit—A hematopoietic cell essential receptor tyrosine kinase. Int. J. Biochem. Cell Biol. 2007; 39:1995–1998. PubMed

Metcalfe D.D.Mast cells and mastocytosis. Blood. 2008; 112:946–956. PubMed PMC

Gregory-Bryson E., Bartlett E., Kiupel M., Hayes S., Yuzbasiyan-Gurkan V.. Canine and human gastrointestinal stromal tumors display similar mutations in c-KIT exon 11. BMC Cancer. 2010; 10:559–568. PubMed PMC

Ashman L.K., Griffith R.. Therapeutic targeting of c-KIT in cancer. Expert Opin. Investig. Drugs. 2013; 22:103–115. PubMed

Lennartsson J., Rönnstrand L.. The stem cell factor receptor/c-Kit as a drug target in cancer. Curr. Cancer Drug Targets. 2006; 6:65–75. PubMed

Fabbro D., Ruetz S., Buchdunger E., Cowan-Jacob S.W., Fendrich G., Liebetanz J., Mestan J., O’Reilly T., Traxler P., Chaudhuri B.et al. .. Protein kinases as targets for anticancer agents: from inhibitors to useful drugs. Pharmacol. Ther. 2002; 93:79–98. PubMed

Rankin S., Reszka A.P., Huppert J., Zloh M., Parkinson G.N., Todd A.K., Ladame S., Balasubramanian S., Neidle S.. Putative DNA quadruplex formation within the human c-kit oncogene. J. Am. Chem. Soc. 2005; 127:10584–10589. PubMed PMC

Yamamoto K., Tojo A., Aoki N., Shibuya M.. Characterization of the promoter region of the human c-kit proto-oncogene. Jpn. J. Cancer Res. 1993; 84:1136–1144. PubMed PMC

Neidle S.Quadruplex Nucleic Acids As Targets For Medicinal Chemistry 1st edition. 2020; Cambridge, USA: Academic Press.

Kuryavyi V., Phan A.T., Patel D.J.. Solution structures of all parallel-stranded monomeric and dimeric G-quadruplex scaffolds of the human c-kit2 promoter. Nucleic Acids Res. 2010; 38:6757–6773. PubMed PMC

Hsu S.T., Varnai P., Bugaut A., Reszka A.P., Neidle S., Balasubramanian S.. A G-rich sequence within the c-kit oncogene promoter forms a parallel G-quadruplex having asymmetric G-tetrad dynamics. J. Am. Chem. Soc. 2009; 131:13399–13409. PubMed PMC

Wei D., Husby J., Neidle S.. Flexibility and structural conservation in a c-KIT G-quadruplex. Nucleic Acids Res. 2015; 43:629–644. PubMed PMC

Wei D., Parkinson G.N., Reszka A.P., Neidle S.. Crystal structure of a c-kit promoter quadruplex reveals the structural role of metal ions and water molecules in maintaining loop conformation. Nucleic Acids Res. 2012; 40:4691–4700. PubMed PMC

Kotar A., Rigo R., Sissi C., Plavec J.. Two-quartet kit* G-quadruplex is formed via double-stranded pre-folded structure. Nucleic Acids Res. 2019; 47:2641–2653. PubMed PMC

Phan A.T., Kuryavyi V., Burge S., Neidle S., Patel D.J.. Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. J. Am. Chem. Soc. 2007; 129:4386–4392. PubMed PMC

Ducani C., Bernardinelli G., Högberg B., Keppler B.K., Terenzi A.. Interplay of Three G-Quadruplex Units in the KIT Promoter. J. Am. Chem. Soc. 2019; 141:10205–10213. PubMed

Rigo R., Sissi C.. Characterization of G4–G4 Crosstalk in the c-KIT Promoter Region. Biochemistry. 2017; 56:4309–4312. PubMed

Raiber E.A., Kranaster R., Lam E., Nikan M., Balasubramanian S.. A non-canonical DNA structure is a binding motif for the transcription factor SP1 in vitro. Nucleic Acids Res. 2012; 40:1499–1508. PubMed PMC

Park G.H., Plummer H.K., Krystal G.W.. Selective Sp1 binding is critical for maximal activity of the human c-kit promoter. Blood. 1998; 92:4138–4149. PubMed

Krasheninina O.A., Novopashina D.S., Apartsin E.K., Venyaminova A.G.. Recent advances in nucleic acid targeting probes and supramolecular constructs based on pyrene-modified oligonucleotides. Molecules. 2017; 22:2108. PubMed PMC

Haider S.M., Neidle S., Parkinson G.N.. A structural analysis of G-quadruplex/ligand interactions. Biochimie. 2011; 93:1239–1251. PubMed

Arola A., Vilar R.. Stabilisation of G-quadruplex DNA by small molecules. Curr. Top. Med. Chem. 2008; 8:1405–1415. PubMed

Asamitsu S., Obata S., Yu Z., Bando T., Sugiyama H.. Recent progress of targeted G-quadruplex-preferred ligands toward cancer therapy. Molecules. 2019; 24:429. PubMed PMC

Yao C., Kraatz H.-B., Steer R.P.. Photophysics of pyrene-labelled compounds of biophysical interest. Photochem. Photobiol. Sci. 2005; 4:191–199. PubMed

Østergaard M.E., Hrdlicka P.J.. Pyrene-functionalized oligonucleotides and locked nucleic acids (LNAs): Tools for fundamental research, diagnostics, and nanotechnology. Chem. Soc. Rev. 2011; 40:5771–5788. PubMed PMC

Astakhova I.K., Pasternak K., Campbell M.A., Gupta P., Wengel J.. A Locked Nucleic Acid-Based Nanocrawler: Designed and Reversible Movement Detected by Multicolor Fluorescence. J. Am. Chem. Soc. 2013; 135:2423–2426. PubMed

Kumar T.S., Myznikova A., Samokhina E., Astakhova I.K.. Rapid genotyping using pyrene−perylene locked nucleic acid complexes. Artif DNA PNA XNA. 2013; 4:58–68. PubMed PMC

Bichenkova E.V., Gbaj A., Walsh L., Savage H.E., Rogert C., Sardarian A.R., Etchells L.L., Douglas K.T.. Detection of nucleic acids in situ: novel oligonucleotide analogues for target-assembled DNA-mounted exciplexes. Org. Biomol. Chem. 2007; 5:1039–1051. PubMed

Bichenkova E.V., Sardarian A.R., Wilton A.N., Bonnet P., Bryce R.A., Douglas K.T.. Exciplex fluorescence emission from simple organic intramolecular constructs in non-polar and highly polar media as model systems for DNA-assembled exciplex detectors. Org. Biomol. Chem. 2006; 4:367–378. PubMed

Bichenkova E.V., Savage H.E., Sardarian A.R., Douglas K.T.. Target-assembled tandem oligonucleotide systems based on exciplexes for detecting DNA mismatches and single nucleotide polymorphisms. Biochem. Biophys. Res. Commun. 2005; 332:956–964. PubMed

Ensslen P., Wagenknecht H.-A.. One-dimensional multichromophor arrays based on DNA: from self-assembly to light-harvesting. Acc. Chem. Res. 2015; 48:2724–2733. PubMed

Teo Y.N., Kool E.T.. DNA-multichromophore systems. Chem. Rev. 2012; 112:4221–4245. PubMed PMC

Filichev V.V., Pedersen E.B.. Stable and selective formation of hoogsteen-type triplexes and duplexes using twisted intercalating nucleic acids (TINA) prepared via postsynthetic sonogashira solid-phase coupling reactions. J. Am. Chem. Soc. 2005; 127:14849–14858. PubMed

Hrdlicka P.J., Kumar T.S., Wengel J.. Targeting of mixed sequence double-stranded DNA using pyrene-functionalized 2′-amino-α-L-LNA. Chem. Commun. 2005; 34:4279–4281. PubMed

Nakamura M., Fukunaga Y., Sasa K., Ohtoshi Y., Kanaori K., Hayashi H., Nakano H., Yamana K.. Pyrene is highly emissive when attached to the RNA duplex but not to the DNA duplex: the structural basis of this difference. Nucleic Acids Res. 2005; 33:5887–5895. PubMed PMC

Lou C., Dallmann A., Marafini P., Gao R., Brown T.. Enhanced H-bonding and π-stacking in DNA: a potent duplex-stabilizing and mismatch sensing nucleobase analogue. Chem. Sci. 2014; 5:3836–3844.

Crescenzo A.D., Ettorre V., Fontana A.. Non-covalent and reversible functionalization of carbon nanotubes. Beilstein J. Nanotechnol. 2014; 5:1675–1690. PubMed PMC

Lemek T., Mazurkiewicz J., Stobinski L., Lin H.M., Tomasik P.. Non-covalent functionalization of multi-walled carbon nanotubes with organic aromatic compounds. J. Nanosci. Nanotechnol. 2007; 7:3081–3088. PubMed

Kovačič M., Podbevšek P., Tateishi-Karimata H., Takahashi S., Sugimoto N., Plavec J.. Thrombin binding aptamer G-quadruplex stabilized by pyrene-modified nucleotides. Nucleic Acids Res. 2020; 48:3975–3986. PubMed PMC

Takahashi S., Kim K.T., Podbevšek P., Plavec J., Kim B.H., Sugimoto N.. Recovery of the formation and function of oxidized G-quadruplexes by a pyrene-modified guanine Tract. J. Am. Chem. Soc. 2018; 140:5774–5783. PubMed

Cogoi S., Zorzet S., Rapozzi V., Géci I., Pedersen E.B., Xodo L.E.. MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice. Nucleic Acids Res. 2013; 41:4049–4064. PubMed PMC

Breslauer K.J.Extracting thermodynamic data from equilibrium melting curves for oligonucleotide order-disorder transitions. Methods in Enzymology, Energetics of Biological Macromolecules. 1995; 259:Cambridge, USA: Academic Press; 221–242. PubMed

Lee W., Tonelli M., Markley J.L.. NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy. Bioinformatics. 2015; 31:1325–1327. PubMed PMC

Zgarbová M., Šponer J., Otyepka M., Cheatham T.E., Galindo-Murillo R., Jurečka P.. Refinement of the sugar–phosphate backbone torsion beta for AMBER force fields improves the description of Z- and B-DNA. J. Chem. Theory Comput. 2015; 11:5723–5736. PubMed

Pérez A., Marchán I., Svozil D., Sponer J., Cheatham T.E., Laughton C.A., Orozco M.. Refinement of the AMBER force field for nucleic acids: improving the description of α/γ conformers. Biophys. J. 2007; 92:3817–3829. PubMed PMC

Krepl M., Zgarbová M., Stadlbauer P., Otyepka M., Banáš P., Koča J., Cheatham T.E., Jurečka P., Šponer J.. Reference simulations of noncanonical nucleic acids with different χ variants of the AMBER force field: quadruplex DNA, quadruplex RNA, and Z-DNA. J. Chem. Theory Comput. 2012; 8:2506–2520. PubMed PMC

Zgarbová M., Luque F.J., Šponer J., Cheatham T.E., Otyepka M., Jurečka P.. Toward improved description of DNA backbone: revisiting epsilon and zeta torsion force field parameters. J. Chem. Theory Comput. 2013; 9:2339–2354. PubMed PMC

Comer J., Gumbart J.C., Hénin J., Lelièvre T., Pohorille A., Chipot C.. The adaptive biasing force method: everything you always wanted to know but were afraid to ask. J. Phys. Chem. B. 2015; 119:1129–1151. PubMed PMC

Minoukadeh K., Chipot C., Lelièvre T.. Potential of mean force calculations: a multiple-walker adaptive biasing force approach. J. Chem. Theory Comput. 2010; 6:1008–1017.

Mones L., Bernstein N., Csányi G.. Exploration, sampling, and reconstruction of free energy surfaces with Gaussian process regression. J. Chem. Theory Comput. 2016; 12:5100–5110. PubMed

Bouchal T., Durník I., Illík V., Réblová K., Kulhánek P.. Importance of base-pair opening for mismatch recognition. Nucleic Acids Res. 2020; 48:11322–11334. PubMed PMC

Humphrey W., Dalke A., Schulten K.. VMD: visual molecular dynamics. J. Mol. Graph. 1996; 14:33–38. PubMed

Fonville J.M., Swart M., Vokáčová Z., Sychrovský V., Šponer J.E., Šponer J., Hilbers C.W., Bickelhaupt F.M., Wijmenga S.S.. Chemical shifts in nucleic acids studied by density functional theory calculations and comparison with experiment. Chem. Eur. J. 2012; 18:12372–12387. PubMed

Fernando H., Reszka A.P., Huppert J., Ladame S., Rankin S., Venkitaraman A.R., Neidle S., Balasubramanian S.. A conserved quadruplex motif located in a transcription activation site of the human c-kit oncogene. Biochemistry. 2006; 45:7854–7860. PubMed PMC

Hou J.-Q., Tan J.-H., Wang X.-X., Chen S.-B., Huang S.-Y., Yan J.-W., Chen S.-H., Ou T.-M., Luo H.-B., Li D.et al. .. Impact of planarity of unfused aromatic molecules on G-quadruplex binding: Learning from isaindigotone derivatives. Org. Biomol. Chem. 2011; 9:6422–6436. PubMed

Ma Y.-Y., Yu S., He X.-J., Xu Y., Wu F., Xia Y.-J., Guo K., Wang H.-J., Ye Z.-Y., Zhang W.et al. .. Involvement of c-KIT mutation in the development of gastrointestinal stromal tumors through proliferation promotion and apoptosis inhibition. Onco Targets Ther. 2014; 7:637–643. PubMed PMC

Yamanoi K., Higuchi K., Kishimoto H., Nishida Y., Nakamura M., Sudoh M., Hirota S.. Multiple gastrointestinal stromal tumors with novel germline c-kit gene mutation, K642T, at exon 13. Hum. Pathol. 2014; 45:884–888. PubMed

Boldrini L., Ursino S., Gisfredi S., Faviana P., Donati V., Camacci T., Lucchi M., Mussi A., Basolo F., Pingitore R.et al. .. Expression and mutational status of c-kit in small-cell lung cancer: prognostic relevance. Clin. Cancer Res. 2004; 10:4101–4108. PubMed

Meng D., Carvajal R.D.. KIT as an oncogenic driver in melanoma: an update on clinical development. Am. J. Clin. Dermatol. 2019; 20:315–323. PubMed

Sakabe T., Azumi J., Haruki T., Umekita Y., Nakamura H., Shiota G.. CD117 expression is a predictive marker for poor prognosis in patients with non-small cell lung cancer. Oncol. Lett. 2017; 13:3703–3708. PubMed PMC

Kee D., Zalcberg J.R.. Current and emerging strategies for the management of imatinib-refractory advanced gastrointestinal stromal tumors. Ther. Adv. Med. Oncol. 2012; 4:255–270. PubMed PMC

Safe S., Abdelrahim M.. Sp transcription factor family and its role in cancer. Eur. J. Cancer. 2005; 41:2438–2448. PubMed