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• W2604003886 abstract "Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5′-AGGGCGGTGTGGGAATAGGGAA-3′) and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5′-AGGGCGGTGTGGGAATAGGGAA-3′) and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets stabilized via eight hydrogen bonds involving both the “Watson-Crick” and “Hoogsteen” edges of each guanine. Stacking of the planar G-quartets (also called a G-tetrad) leads to the formation of a G-quadruplex (G4). 3The abbreviations used are: G4G-quadruplexNHEnuclease-hypersensitive elementMAZMyc-associated zinc fingerDMSdimethyl sulfateDOSYdiffusion-ordered spectroscopyr.m.s.d.root mean square deviationdABzN6-benzoyl-2′-deoxyadenosinedCAcN4-acetyl-2′-deoxycytidinedGiBuN2-isobutyryl-2′-deoxyguanosineHSQCheteronuclear single quantum coherenceTOCSYtotal correlation spectroscopyHMBCheteronuclear multiple bond correlation. G4s are maintained by the presence of cations such as K+ and to a lesser degree Na+ and NH4+. The stacked G-quartets constitute the nearly invariant core of all G4 structures (1Balasubramanian S. Neidle S. G-quadruplex nucleic acids as therapeutic targets.Curr. Opin. Chemical Biol. 2009; 13: 345-353Crossref PubMed Scopus (470) Google Scholar, 2Düchler M. G-quadruplexes: targets and tools in anticancer drug design.J. Drug Target. 2012; 20: 389-400Crossref PubMed Scopus (88) Google Scholar3Phan A.T. Human telomeric G-quadruplex: structures of DNA and RNA sequences.FEBS J. 2010; 277: 1107-1117Crossref PubMed Scopus (415) Google Scholar). This core is stabilized by cooperation between three key factors: hydrogen-bonding dipole interactions, metal coordination, and π-π stacking. The orientations of the loop regions within G4 structures are tightly related to strand directionality and give rise to the heterogeneity of G4 structures (4Wong A. Ida R. Spindler L. Wu G. Disodium guanosine 5′-monophosphate self-associates into nanoscale cylinders at pH 8: a combined diffusion NMR spectroscopy and dynamic light scattering study.J. Am. Chem. Soc. 2005; 127: 6990-6998Crossref PubMed Scopus (119) Google Scholar, 5Phan A.T. Kuryavyi V. Luu K.N. Patel D.J. Structure of two intramolecular G-quadruplexes formed by natural human telomere sequences in K+ solution.Nucleic Acids Res. 2007; 35: 6517-6525Crossref PubMed Scopus (418) Google Scholar6Marusic M. Sket P. Bauer L. Viglasky V. Plavec J. Solution-state structure of an intramolecular G-quadruplex with propeller, diagonal and edgewise loops.Nucleic Acids Res. 2012; 40: 6946-6956Crossref PubMed Scopus (61) Google Scholar). G4 structures interfere with replication (7Madireddy A. Purushothaman P. Loosbroock C.P. Robertson E.S. Schildkraut C.L. Verma S.C. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.Nucleic Acids Res. 2016; 44: 3675-3694Crossref PubMed Scopus (57) Google Scholar, 8Valton A.L. Prioleau M.N. G-quadruplexes in DNA replication: a problem or a necessity?.Trends Genet. 2016; 32: 697-706Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar9Lopes J. Piazza A. Bermejo R. Kriegsman B. Colosio A. Teulade-Fichou M.P. Foiani M. Nicolas A. G-quadruplex-induced instability during leading-strand replication.EMBO J. 2011; 30: 4033-4046Crossref PubMed Scopus (227) Google Scholar), transcription (10Du Z. Kong P. Gao Y. Li N. Enrichment of G4 DNA motif in transcriptional regulatory region of chicken genome.Biochem. Biophys. Res. Commun. 2007; 354: 1067-1070Crossref PubMed Scopus (35) Google Scholar11Yan J. Zhao X. Liu B. Yuan Y. Guan Y. An intramolecular G-quadruplex structure formed in the human MET promoter region and its biological relevance.Mol. Carcinog. 2016; 55: 897-909Crossref PubMed Scopus (16) Google Scholar, 12Cogoi S. Xodo L.E. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription.Nucleic Acids Res. 2006; 34: 2536-2549Crossref PubMed Scopus (553) Google Scholar13Cogoi S. Paramasivam M. Membrino A. Yokoyama K.K. Xodo L.E. The KRAS promoter responds to Myc-associated zinc finger and poly(ADP-ribose) polymerase 1 proteins, which recognize a critical quadruplex-forming GA-element.J. Biol. Chem. 2010; 285: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and recombination (14Stanton A. Harris L.M. Graham G. Merrick C.J. Recombination events among virulence genes in malaria parasites are associated with G-quadruplex-forming DNA motifs.BMC Genomics. 2016; 17: 859Crossref PubMed Scopus (28) Google Scholar, 15Piekna-Przybylska D. Sullivan M.A. Sharma G. Bambara R.A. U3 region in the HIV-1 genome adopts a G-quadruplex structure in its RNA and DNA sequence.Biochemistry. 2014; 53: 2581-2593Crossref PubMed Scopus (68) Google Scholar16De Nicola B. Lech C.J. Heddi B. Regmi S. Frasson I. Perrone R. Richter S.N. Phan A.T. Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.Nucleic Acids Res. 2016; 44: 6442-6451Crossref PubMed Scopus (51) Google Scholar). Bioinformatics analyses have provided evidence that sequences with potential to adopt G4 are not randomly localized within genomes but are specifically enriched in particular regions such as telomeres and promoters of genes (17Huppert J.L. Balasubramanian S. Prevalence of quadruplexes in the human genome.Nucleic Acids Res. 2005; 33: 2908-2916Crossref PubMed Scopus (1284) Google Scholar18Wong H.M. Stegle O. Rodgers S. Huppert J.L. A toolbox for predicting G-quadruplex formation and stability.J. Nucleic Acids. 2010; 2010564946Crossref PubMed Scopus (51) Google Scholar, 19Todd A.K. Johnston M. Neidle S. Highly prevalent putative quadruplex sequence motifs in human DNA.Nucleic Acids Res. 2005; 33: 2901-2907Crossref PubMed Scopus (779) Google Scholar20Stegle O. Payet L. Mergny J.L. MacKay D.J. Leon J.H. Predicting and understanding the stability of G-quadruplexes.Bioinformatics. 2009; 25: i374-i382Crossref PubMed Scopus (82) Google Scholar). Proto-oncogenes are particularly enriched with G4 motifs, whereas tumor suppressor genes are not (21Huppert J.L. Balasubramanian S. G-quadruplexes in promoters throughout the human genome.Nucleic Acids Res. 2007; 35: 406-413Crossref PubMed Scopus (959) Google Scholar, 22Eddy J. Maizels N. Gene function correlates with potential for G4 DNA formation in the human genome.Nucleic Acids Res. 2006; 34: 3887-3896Crossref PubMed Scopus (394) Google Scholar). The formation of intramolecular G4s has been studied in vitro for motifs found in different human promoters regions, including c-myc (23Ambrus A. Chen D. Dai J. Jones R.A. Yang D. Solution structure of the biologically relevant G-quadruplex element in the human c-MYC promoter. Implications for G-quadruplex stabilization.Biochemistry. 2005; 44: 2048-2058Crossref PubMed Scopus (491) Google Scholar, 24Phan A.T. Kuryavyi V. Gaw H.Y. Patel D.J. Small-molecule interaction with a five-guanine-tract G-quadruplex structure from the human MYC promoter.Nat. Chem. Biol. 2005; 1: 167-173Crossref PubMed Scopus (432) Google Scholar25Phan A.T. Modi Y.S. Patel D.J. Propeller-type parallel-stranded G-quadruplexes in the human c-myc promoter.J. Am. Chem. Soc. 2004; 126: 8710-8716Crossref PubMed Scopus (445) Google Scholar), c-kit (26Phan 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: 4386-4392Crossref PubMed Scopus (367) Google Scholar), and bcl2 (27Dai J. Dexheimer T.S. Chen D. Carver M. Ambrus A. Jones R.A. Yang D. An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution.J. Am. Chem. Soc. 2006; 128: 1096-1098Crossref PubMed Scopus (342) Google Scholar). G-rich elements found in other proto-oncogenes such as KRAS have received less attention. KRAS is located on chromosome 12 and encodes a GTP/GDP-binding protein. Previous studies showed that mutant alleles of KRAS are prevalent in pancreatic, biliary tract, colorectal, and lung carcinomas (28Lavrado J. Brito H. Borralho P.M. Ohnmacht S.A. Kim N.S. Leitão C. Pisco S. Gunaratnam M. Rodrigues C.M. Moreira R. Neidle S. Paulo A. KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2-c]quinolines.Sci. Rep. 2015; 59696Crossref PubMed Scopus (71) Google Scholar29Brito H. Martins A.C. Lavrado J. Mendes E. Francisco A.P. Santos S.A. Ohnmacht S.A. Kim N.S. Rodrigues C.M. Moreira R. Neidle S. Borralho P.M. Paulo A. Targeting KRAS oncogene in colon cancer cells with 7-carboxylate indolo[3,2-b]quinoline tri-alkylamine derivatives.PLoS One. 2015; 10e0126891Crossref PubMed Scopus (42) Google Scholar, 30Forbes S.A. Bindal N. Bamford S. Cole C. Kok C.Y. Beare D. Jia M. Shepherd R. Leung K. Menzies A. Teague J.W. Campbell P.J. Stratton M.R. Futreal P.A. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer.Nucleic Acids Res. 2011; 39: D945-D950Crossref PubMed Scopus (1812) Google Scholar, 31Dent P. Multi-kinase modulation for colon cancer therapy.Cancer Biol. Ther. 2013; 14: 877-878Crossref PubMed Scopus (6) Google Scholar, 32Krens L.L. Baas J.M. Gelderblom H. Guchelaar H.J. Therapeutic modulation of k-ras signaling in colorectal cancer.Drug Discov. Today. 2010; 15: 502-516Crossref PubMed Scopus (33) Google Scholar33Andreyev H.J. Ross P.J. Cunningham D. Clarke P.A. Antisense treatment directed against mutated Ki-ras in human colorectal adenocarcinoma.Gut. 2001; 48: 230-237Crossref PubMed Scopus (20) Google Scholar). Mutations in the KRAS promoter are found in about 30% of these cases. It is thought that the KRAS oncogene promotes glycolysis through the activation of downstream signaling pathways (34Ying H. Kimmelman A.C. Lyssiotis C.A. Hua S. Chu G.C. Fletcher-Sananikone E. Locasale J.W. Son J. Zhang H. Coloff J.L. Yan H. Wang W. Chen S. Viale A. Zheng H. et al.Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.Cell. 2012; 149: 656-670Abstract Full Text Full Text PDF PubMed Scopus (1279) Google Scholar) to sustain the energy requirements for uncontrolled cellular proliferation, thus contributing to survival of cancer cells. The very high affinity of the RAS GTP/GDP-binding site (picomolar range) (35John J. Sohmen R. Feuerstein J. Linke R. Wittinghofer A. Goody R.S. Kinetics of interaction of nucleotides with nucleotide-free H-ras p21.Biochemistry. 1990; 29: 6058-6065Crossref PubMed Scopus (346) Google Scholar, 36McCormick F. K-Ras protein as a drug target.J. Mol. Med. 2016; 94: 253-258Crossref PubMed Scopus (74) Google Scholar) has made it difficult to synthesize molecules that effectively compete with GTP at millimolar range inside cells to block KRAS activity (37Cox A.D. Fesik S.W. Kimmelman A.C. Luo J. Der C.J. Drugging the undruggable RAS: mission possible?.Nat. Rev. Drug Discov. 2014; 13: 828-851Crossref PubMed Scopus (1181) Google Scholar). It is no surprise that after many decades of unsuccessfully battling against the RAS proteins new strategies in the field of drug design have emerged. Among those, some target alternative binding sites in the GTPase domain (38Ostrem J.M. Peters U. Sos M.L. Wells J.A. Shokat K.M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions.Nature. 2013; 503: 548-551Crossref PubMed Scopus (1272) Google Scholar), others target the altered metabolic pathways (39Zhu Z. Golay H.G. Barbie D.A. Targeting pathways downstream of KRAS in lung adenocarcinoma.Pharmacogenomics. 2014; 15: 1507-1518Crossref PubMed Scopus (24) Google Scholar), whereas some target the mRNA with antisense oligonucleotides (40Wang J.H. Newbury L.J. Knisely A.S. Monia B. Hendry B.M. Sharpe C.C. Antisense knockdown of Kras inhibits fibrosis in a rat model of unilateral ureteric obstruction.Am. J. Pathol. 2012; 180: 82-90Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Alternatively, several strategies, including ours, target the promotor region in an effort to block KRAS expression (41Cogoi S. Xodo L.E. G4 DNA in ras genes and its potential in cancer therapy.Biochim. Biophys. Acta. 2016; 1859: 663-674Crossref PubMed Scopus (40) Google Scholar). The KRAS promoter contains a polypurine nuclease-hypersensitive element (42Rokney A. Shagan M. Kessel M. Smith Y. Rosenshine I. Oppenheim A.B. E. coli transports aggregated proteins to the poles by a specific and energy-dependent process.J. Mol. Biol. 2009; 392: 589-601Crossref PubMed Scopus (86) Google Scholar) that plays an essential role in transcription. Its deletion results in a significant down-regulation of KRAS transcription (12Cogoi S. Xodo L.E. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription.Nucleic Acids Res. 2006; 34: 2536-2549Crossref PubMed Scopus (553) Google Scholar, 43Cogoi S. Paramasivam M. Spolaore B. Xodo L.E. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins.Nucleic Acids Res. 2008; 36: 3765-3780Crossref PubMed Scopus (123) Google Scholar, 44Paramasivam M. Membrino A. Cogoi S. Fukuda H. Nakagama H. Xodo L.E. Protein hnRNP A1 and its derivative Up1 unfold quadruplex DNA in the human KRAS promoter: implications for transcription.Nucleic Acids Res. 2009; 37: 2841-2853Crossref PubMed Scopus (116) Google Scholar). The promoter region of the KRAS gene comprises more than 500 bp and is susceptible to digestion by nucleases such as DNase I, micrococcal nuclease, and other endogenous nucleases (45Jordano J. Perucho M. Chromatin structure of the promoter region of the human c-K-ras gene.Nucleic Acids Res. 1986; 14: 7361-7378Crossref PubMed Scopus (31) Google Scholar). A particular sequence inside the NHE, between positions −327 and −296 nucleotides upstream of the main transcription initiation site, is particularly rich in guanines (sequence 32R; Fig. 1A). 32R contains six guanine stretches and is able to form several G4 conformations. Among those stretches of guanines, two regions overlap and includes the Myc-associated zinc finger (MAZ)-binding sites, the recognition sequence for a transcription factor that recognizes GGGCGG and GGGAGG sequences (46Cogoi 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: 4049-4064Crossref PubMed Scopus (79) Google Scholar). Recent biophysical studies using circular dichroism and DMS footprinting (43Cogoi S. Paramasivam M. Spolaore B. Xodo L.E. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins.Nucleic Acids Res. 2008; 36: 3765-3780Crossref PubMed Scopus (123) Google Scholar, 47Cogoi S. Paramasivam M. Filichev V. Géci I. Pedersen E.B. Xodo L.E. Identification of a new G-quadruplex motif in the KRAS promoter and design of pyrene-modified G4-decoys with antiproliferative activity in pancreatic cancer cells.J. Med. Chem. 2009; 52: 564-568Crossref PubMed Scopus (74) Google Scholar) suggest that oligonucleotides corresponding to 32R are able to adopt different intramolecular G-quadruplex topologies depending on which G-runs are included. Some of the topologies were tested as decoys for sequestration of MAZ (48Podbevšek P. Plavec J. KRAS promoter oligonucleotide with decoy activity dimerizes into a unique topology consisting of two G-quadruplex units.Nucleic Acids Res. 2016; 44: 917-925Crossref PubMed Scopus (21) Google Scholar). In addition, certain G4 structures in this region are stabilized by G-quadruplex-interacting ligands (12Cogoi S. Xodo L.E. G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription.Nucleic Acids Res. 2006; 34: 2536-2549Crossref PubMed Scopus (553) Google Scholar, 13Cogoi S. Paramasivam M. Membrino A. Yokoyama K.K. Xodo L.E. The KRAS promoter responds to Myc-associated zinc finger and poly(ADP-ribose) polymerase 1 proteins, which recognize a critical quadruplex-forming GA-element.J. Biol. Chem. 2010; 285: 22003-22016Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 43Cogoi S. Paramasivam M. Spolaore B. Xodo L.E. Structural polymorphism within a regulatory element of the human KRAS promoter: formation of G4-DNA recognized by nuclear proteins.Nucleic Acids Res. 2008; 36: 3765-3780Crossref PubMed Scopus (123) Google Scholar, 49Xodo L. Paramasivam M. Membrino A. Cogoi S. Protein hnRNPA1 binds to a critical G-rich element of KRAS and unwinds G-quadruplex structures: implications in transcription.Nucleic Acids Symp. Ser. 2008; 52: 159-160Crossref Google Scholar) such as guanidine-modified phthalocyanines that interfere with KRAS transcription by competing with MAZ and poly(ADP-ribose) polymerase 1 proteins. Here we studied the conformations adopted by different stretches within the 32R sequence using NMR spectroscopy. The G4 conformation revealed by our studies provides a model that could potentially be used for in silico drug screening for ligands that stabilize the G4 structure in the KRAS promoter. The approach targeting unusual motifs present in genomic DNA is actively being pursued and can be seen as a new alternative strategy with promising results (1Balasubramanian S. Neidle S. G-quadruplex nucleic acids as therapeutic targets.Curr. Opin. Chemical Biol. 2009; 13: 345-353Crossref PubMed Scopus (470) Google Scholar, 28Lavrado J. Brito H. Borralho P.M. Ohnmacht S.A. Kim N.S. Leitão C. Pisco S. Gunaratnam M. Rodrigues C.M. Moreira R. Neidle S. Paulo A. KRAS oncogene repression in colon cancer cell lines by G-quadruplex binding indolo[3,2-c]quinolines.Sci. Rep. 2015; 59696Crossref PubMed Scopus (71) Google Scholar, 29Brito H. Martins A.C. Lavrado J. Mendes E. Francisco A.P. Santos S.A. Ohnmacht S.A. Kim N.S. Rodrigues C.M. Moreira R. Neidle S. Borralho P.M. Paulo A. Targeting KRAS oncogene in colon cancer cells with 7-carboxylate indolo[3,2-b]quinoline tri-alkylamine derivatives.PLoS One. 2015; 10e0126891Crossref PubMed Scopus (42) Google Scholar, 41Cogoi S. Xodo L.E. G4 DNA in ras genes and its potential in cancer therapy.Biochim. Biophys. Acta. 2016; 1859: 663-674Crossref PubMed Scopus (40) Google Scholar). G-quadruplex nuclease-hypersensitive element Myc-associated zinc finger dimethyl sulfate diffusion-ordered spectroscopy root mean square deviation N6-benzoyl-2′-deoxyadenosine N4-acetyl-2′-deoxycytidine N2-isobutyryl-2′-deoxyguanosine heteronuclear single quantum coherence total correlation spectroscopy heteronuclear multiple bond correlation. We began our study with circular dichroism (CD) and NMR analyses of oligonucleotides within the sequence NHE (Fig. 1A). To make spectral assignments possible, different oligonucleotides were evaluated (supplemental Table S2) with the objective of identifying a sequence that formed a single G4 conformer based on the dispersion and intensities of imino peaks observed in the NMR spectra. The sequence 21R and three other related sequences display a 1D imino peak pattern that corresponds to a single conformer as shown by 1D 1H NMR spectroscopy (Fig. 1C). The similar imino signatures suggest the presence of a predominant conformer within the human NHE of KRAS gene. The sequence 22RT with a G16 to T16 mutation displayed a better resolved imino peak pattern and a slightly better stabilization observed by the CD melting studies (Fig. 1B). In addition, DMS footprinting (50Paramasivam M. Cogoi S. Xodo L.E. Primer extension reactions as a tool to uncover folding motifs within complex G-rich sequences: analysis of the human KRAS NHE.Chem. Commun. 2011; 47: 4965-4967Crossref PubMed Scopus (24) Google Scholar) and our 15N-filtered 1D NMR experiments (results not shown) demonstrated that G16 did not participate in the tetrad formation. Oligonucleotides of the native sequence with four G-tracts, 21R and 22R, and those with single G16 to T (16G→T) mutations, 21RT and 22RT, respectively, appeared to adopt a predominant conformation based on analysis of the imino proton region from 10 to 12 ppm. The sequence and the respective 1H 1D NMR spectra are presented in Fig. 1C. Remarkably, 22RT showed a better resolved peak pattern in both imino and aromatic regions (not shown) than did 21R or 22R. For stability purposes, we have included an additional A at the 22RT 3′-end. These modifications resulted in better imino and aromatic peak resolution when compared with 22R and 21R. The CD spectral signatures are similar between 21R and 22RT. The spectrum of each includes a positive band at 263 nm and a negative band at 243 nm suggestive of a parallel G4 fold (Fig. 1B, left). The thermal stability of 21R and 22RT was determined through CD melting experiments. The melting temperatures (Tm) in 90 mm K+ were 49.2 ± 0.2 and 51.8 ± 0.3 °C for 21R and 22RT, respectively (Fig. 1B, right). The molecularity of 21R and 22RT was assessed by inspecting the UV-visible melting curves and by diffusion NMR experiments (supplemental Figs. S2 and S3, respectively). The results demonstrate that both 21R and 22RT fold into a monomeric structure, inferred from the reversible and superimposable cooling versus heating UV-visible curves in the concentration range that spans an order of magnitude from ≈5 to ≈50 μm. These experiments showed that melting transitions are reversible and independent of DNA concentration, demonstrating that the G4 structures formed by both 21R and 22RT are unimolecular. As the melting process was reversible, model-dependent van't Hoff enthalpies of folding could be calculated. The ΔH037 values for 21R and 22RT were 164 ± 4 and 191 ± 7 kJ/mol, respectively. Diffusion NMR spectroscopy was used to determine the diffusion coefficient value for 22RT. From diffusion-ordered spectroscopy (DOSY) experiments, we obtained a diffusion coefficient of 1.54 × 10−10 m2 s−1 (logD ≈ −9.81), which is in the range of those found for monomeric G4 oligonucleotides of similar size such as the human telomeric sequence (22AG) observed elsewhere (23Ambrus A. Chen D. Dai J. Jones R.A. Yang D. Solution structure of the biologically relevant G-quadruplex element in the human c-MYC promoter. Implications for G-quadruplex stabilization.Biochemistry. 2005; 44: 2048-2058Crossref PubMed Scopus (491) Google Scholar, 51Kerkour A. Mergny J.L. Salgado G.F. NMR based model of human telomeric repeat G-quadruplex in complex with 2,4,6-triarylpyridine family ligand.Biochim. Biophys. Acta. 2016; 10.1016/j.bbagen.2016.12.016Google Scholar, 52Groves P. Webba da Silva M. Rapid stoichiometric analysis of G-quadruplexes in solution.Chemistry. 2010; 16: 6451-6453Crossref PubMed Scopus (7) Google Scholar). The results support a model where 22RT is monomeric under the experimental conditions probed in this work. For reference, we report a diffusion value of 8.9 × 10−11 m2 s−1 (logD ≈ −10.05) obtained for the oligonucleotide KRAS 44R from the NHE region that contains the sequence 32R at the 5′-end (supplemental Table S2). The imino proton spectrum of 22RT is characterized by 10 individually well resolved and sharp peaks in the 10.5-12-ppm region (Fig. 1C) plus one additional broad peak that was later identified as G19 and G7 overlapped imino peaks. The imino pattern in this region is often used as a fingerprint for G4 structures. The pattern observed suggests the formation of three G-quartets, each involving four imino protons. Based on the specific intraquartet characteristic guanine H1–H8 NOE correlations (Fig. 2B), the folding pattern of the 22RT G4 involves three G-quartets: G2·G6·G11·G18, G3·G7·G12·G19, and G4·G9·G13·G20 (Fig. 2). For clarity, we have selected the six lowest-energy structures after refinement of the best 20 structures with a heavy atom r.m.s.d. value of ≈1.5 Å (Fig. 3). When depicted against the calculated mass-weighting principal axis, the tetrad core is placed with its averaged planes almost in a perpendicular fashion (Fig. 3). The G3·G7·G12·G19 residues form the central tetrad of the quadruplex core as the imino protons on these residues are better protected from water/deuterium exchange than are those of other guanines of 22RT (supplemental Fig. S4). In addition and as expected, the guanines in a central G-quartet have by far the strongest NOE inter-residue connectivities between exchangeable protons (e.g. NH2/H1), supporting the increased protection of these protons from exchange with water. Interestingly, G3 is the only base that has NOE cross-peaks to both amino-exchangeable protons (NH21/NH22). These protons are probably protected by the single-nucleotide chain reversal C5 loop, which bridges and completely blocks the groove between G3 and G7. The 22RT G4 has an ˜30° helical twist on average and a rise of 3.4 Å for each G-tetrad step. On average, the four grooves are of medium size with similar widths in the range of 12 ± 2 Å as defined by the distances between phosphates of opposing guanines in the structure. The orientations of the aromatic bases toward the sugars are determined by the conformation of the glycosidic bond angle, which is determined by the intraresidue NOE correlation intensities between the H8 aromatic proton and H1′ sugar proton. All the guanine glycosidic torsion angles are in the anti conformation as reflected by the medium/low intraguanine NOE cross-peaks observed between H8 and H1′ protons (Fig. 2A). These glycosidic torsion angle conformations are expected for a parallel G4 as suggested by the CD spectra of 22RT (Fig. 1B). Our CD and NMR results are consistent and indicate that 22RT adopts a parallel G4 as shown schematically in Fig. 2C. The three G-tetrads are connected by four linkers: two single-residue loops (C5 and T10), a bulge (T8), and one four-nucleotide loop (A14, A15, T16, and A17). C5 and T10 each form double-chain reversal loops that allow these single residues to bridge three G-tetrad blocks. Inspection of r.m.s.d. values and conformation diversity for C5 indicates hingelike motions parallel to the mass-weighting principal axis. The T10 base is oriented toward the 3′-end of the oligonucleotides, and fewer distinct conformers are observed. Interestingly, T8 forms a bulge projected out of the G-tetrad core. Although over 700,000 G-quadruplexes with single or multiple bulges may exist in the human genome (53Chambers 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: 877-881Crossref PubMed Scopus (702) Google Scholar, 54Bedrat A. Lacroi" @default.
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• W2604003886 date "2017-05-01" @default.
• W2604003886 modified "2023-10-18" @default.
• W2604003886 title "High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation" @default.
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• W2604003886 doi "https://doi.org/10.1074/jbc.m117.781906" @default.
• W2604003886 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5427283" @default.
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