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• W2040576048 abstract "Triplex-forming oligonucleotides (TFOs), as DNA-binding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine·oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect. Triplex-forming oligonucleotides (TFOs), as DNA-binding molecules that recognize specific sequences, offer unique potential for the understanding of processes occurring on DNA and associated functions. They are also powerful DNA recognition elements for the positioning of ubiquitous molecules acting on DNA, such as anticancer drugs. A prerequisite for further development of DNA code-reading molecules including TFOs is their ability to form a complex in a cellular context: their binding affinities must be comparable to those of DNA-associated proteins. To reach this goal, chemically modified TFOs must be developed. In this work, we present triplex-forming properties (kinetics and thermodynamics) and cellular activity of G-containing locked nucleic acid-modified TFOs (TFO/LNAs). In conditions simulating physiological ones, these TFO/LNAs strongly enhanced triplex stability compared with the non-modified TFO or with the pyrimidine TFO/LNA directed against the same oligopyrimidine·oligopurine sequence, mainly by decreasing the dissociation rate constant and conferring an entropic gain. We provide evidence of their biological activity by a triplex-based mechanism, in vitro and in a cellular context, under conditions in which the parent phosphodiester oligonucleotide did not exhibit any inhibitory effect. The design of synthetic non-protein molecules able to control DNA-associated biological functions through their interaction with a specific DNA sequence represents a very attractive approach. Among DNA code-reading molecules, triplex-forming oligonucleotides (TFOs) 1The abbreviations used are: TFO, triplex-forming oligonucleotide; LNA, locked nucleic acid; TFO/LNA, LNA-modified triplex-forming oligonucleotide; nt, nucleotide(s); GFP, green fluorescent protein; CMV, cytomegalovirus; PPT, polypurine tract; nt, nucleotide(s); SPR, surface plasmon resonance. are able to bind to the major groove of oligopyrimidine·oligopurine regions in double-stranded DNA. A series of results have validated triplex-based approaches at the molecular and cellular levels: triplexes have been shown to interfere with transcription (initiation and elongation), replication, repair, and recombination (1Besch R. Giovannangeli C. Degitz K. Curr. Drug Targets. 2004; 5: 691-703Crossref PubMed Scopus (54) Google Scholar, 2Seidman M.M. Glazer P.M. J. Clin. Investig. 2003; 112: 487-494Crossref PubMed Scopus (144) Google Scholar). However, the intracellular efficiency of TFOs still has to be improved. One possible approach consists of increasing the stability of the non-covalent triple helices under physiological conditions to reach binding affinities comparable to those of DNA-associated regulatory proteins. To this end, a variety of chemically modified nucleic acids have been developed. Among them are locked nucleic acids (LNAs) that contain LNA nucleotide monomers, i.e. ribonucleotides with a 2′-O,4′-C-methylene linkage that effects conformational fixation of the furanose ring in a C3′-endo conformation (3Koshkin A. Rajwanshi V.K. Wengel J. Tetrahedron Lett. 1998; 39: 4381-4384Crossref Scopus (122) Google Scholar, 4Obika S. Nanbu D. Hari Y. Andoh J. Morio K. Doi T. Imanishi T. Tetrahedron Lett. 1998; 39: 5401-5404Crossref Scopus (615) Google Scholar). LNA-containing oligonucleotides have been recently shown to enhance triplex stability and to alleviate in part the sequence constraints imposed by the triple helical recognition motifs (5Buchini S. Leumann C.J. Curr. Opin. Chem. Biol. 2003; 7: 717-726Crossref PubMed Scopus (147) Google Scholar, 6Wengel J. Vester B. Lundberg L.B. Douthwaite S. Sorensen M.D. Babu B.R. Gait M.J. Arzumanov A. Petersen M. Nielsen J.T. Nucleosides Nucleotides Nucleic Acids. 2003; 22: 601-604Crossref PubMed Scopus (22) Google Scholar). Only a few hybridization properties of LNA-modified TFOs (TFO/LNAs) have been reported so far, and they all concern (T,C)-containing TFO/LNAs. It has been shown that fully modified TFO/LNAs failed to bind to double-stranded DNA (7Obika S. Uneda T. Sugimoto T. Nanbu D. Minami T. Doi T. Imanishi T. Bioorg. Med. Chem. 2001; 9: 1001-1011Crossref PubMed Scopus (151) Google Scholar, 8Koizumi M. Morita K. Daigo M. Tsutsumi S. Abe K. Obika S. Imanishi T. Nucleic Acids Res. 2003; 31: 3267-3273Crossref PubMed Scopus (55) Google Scholar), likely due to conformational restraint of TFO/LNA. However, alternating DNA and LNA nucleotides in TFO sequences is appropriate for efficient triplex formation. A study on a 15-mer pyrimidine TFO/LNA provided evidence that LNA-induced triplex stabilization is associated with a slower dissociation rate constant and a less unfavorable entropic contribution compared with the non-modified TFO (9Torigoe H. Hari Y. Sekiguchi M. Obika S. Imanishi T. J. Biol. Chem. 2001; 276: 2354-2360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In previous works, TFO/LNAs have been used only for in vitro assays, including plasmid functionalization (10Hertoghs K.M. Ellis J.H. Catchpole I.R. Nucleic Acids Res. 2003; 31: 5817-5830Crossref PubMed Scopus (37) Google Scholar). However, LNA-modified oligonucleotides are appealing molecules: they have been successfully used as efficient antisense agents, even in vivo (for examples, see Refs. 11Elayadi A.N. Braasch D.A. Corey D.R. Biochemistry. 2002; 41: 9973-9981Crossref PubMed Scopus (92) Google Scholar, 12Nulf C.J. Corey D. Nucleic Acids Res. 2004; 32: 3792-3798Crossref PubMed Scopus (71) Google Scholar, 13Fluiter K. ten Asbroek A.L. de Wissel M.B. Jakobs M.E. Wissenbach M. Olsson H. Olsen O. Oerum H. Baas F. Nucleic Acids Res. 2003; 31: 953-962Crossref PubMed Scopus (150) Google Scholar), and also as decoys (14Crinelli R. Bianchi M. Gentilini L. Palma L. Sorensen M.D. Bryld T. Babu R.B. Arar K. Wengel J. Magnani M. Nucleic Acids Res. 2004; 32: 1874-1885Crossref PubMed Scopus (40) Google Scholar), aptamers (15Schmidt K.S. Borkowski S. Kurreck J. Stephens A.W. Bald R. Hecht M. Friebe M. Dinkelborg L. Erdmann V.A. Nucleic Acids Res. 2004; 32: 5757-5765Crossref PubMed Scopus (248) Google Scholar), LNAzymes (16Vester B. Lundberg L.B. Sorensen M.D. Babu B.R. Douthwaite S. Wengel J. J. Am. Chem. Soc. 2002; 124: 13682-13683Crossref PubMed Scopus (113) Google Scholar), and DNA-correcting agents (17Parekh-Olmedo H. Drury M. Kmiec E.B. Chem. Biol. 2002; 9: 1073-1084Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In the present work we report the triplex forming properties and intracellular activity of G-containing TFO/LNAs. We characterized the mechanism of triplex stabilization induced by LNA modifications in G-containing TFOs, compared with non-modified isosequential phosphodiester TFOs and with pyrimidine TFO/LNA directed against the same oligopyrimidine· oligopurine target sequence. The dependence of triplex stability on pH was also explored. To address these questions, kinetic and thermodynamic parameters of triplexes were evaluated by surface plasmon resonance. Specificity of TFO/LNA binding was also analyzed, using UV melting experiments, electrophoretic mobility shift assays, and restriction enzymatic protection assay. Finally, we evaluated the capacity of G-containing TFO/LNAs to interfere with biological processes in vitro and in cells, using experimental settings designed to demonstrate a triplex-based mechanism in a quantitative manner. We showed that G-containing TFO/LNAs were active, as measured by inhibition of transcription elongation. Our results support TFO/LNA activity in a cellular context at submicromolar concentrations, which has never been reported before, and represent the first step toward further development of TFO/LNAs as artificial modulators of DNA-associated biological functions. Oligonucleotides—Oligonucleotide analogues with LNA residues were synthesized as previously described (3Koshkin A. Rajwanshi V.K. Wengel J. Tetrahedron Lett. 1998; 39: 4381-4384Crossref Scopus (122) Google Scholar) or purchased from Proligo (France SAS). Phopodiester oligonucleotides were synthesized by Eurogentec; for cellular experiments, the phosphodiester oligonucleotide (15TCG sequence, see Fig. 1) was 3′-modified by incorporation of a propylamine group (named 15TCG*/po) to resist nuclease-mediated degradation. Plasmids—The pSP-F47 plasmid was constructed by insertion of a 780-bp fragment of the human immunodeficiency virus type 1/nef gene containing the oligopyrimidine·oligopurine PPT target sequence between the T7 and SP6 promoters in the pSP73 host vector (Promega), as previously described in detail (18Giovannangeli C. Perrouault L. Escude C. Gryaznov S. Helene C. J. Mol. Biol. 1996; 261: 386-398Crossref PubMed Scopus (59) Google Scholar). This system allowed us to run bidirectional in vitro transcription assays. The pCMV(+)PPT/luc plasmid derives from the bidirectional expression vectors (pBI Tet vectors; Clontech). These vectors contain the tet-responsive element between two identical minimal CMV promoters in opposite directions. This system was used to express two reporter genes, the firefly luciferase (Photinus pyralis) gene (luc) and the GFP gene. Expression of both genes is co-regulated by doxycycline. The activator protein (reverse Tet repressor protein, rTetR) binds the tetresponsive element in the presence of doxycycline and activates transcription. This protein was produced from the pTet-On expression plasmid (Clontech). Two 55-bp inserts containing either the wild-type human immunodeficiency virus type 1 polypurine tract target sequence (PPT, 5′-AAAAGAAAAGGGGGGA-3′) or a mutated sequence (PPTmut, 5′-AAAAGAAGGGGAGGAA-3′; the four mutations are shown in bold) were cloned in the 5′-transcribed but untranslated regions of the luciferase and GFP genes, downstream of the transcription start site. The pRL-CMV vector (Promega) contains Renilla luciferase (from the marine organism Renilla reniformis) under the control of the CMV promoter. pRL-CMV was used to monitor transfection efficiency. UV Absorption Melting Experiments—All thermal denaturation experiments were performed in a 10 mm sodium cacodylate buffer (at the indicated pH value) containing 10 mm MgCl2 and 150 mm NaCl. The sample contained 1 μm duplex (PPT or mutPPT duplex: 29-bp-long intramolecular hairpin duplexes) and 1.5 μm TFO (see the sequences in Fig. 1). Melting profiles were recorded at 260 nm, using an UVIKON 940 spectrophometer, as previously described (19Faria M. Wood C. White M. Helen C. Giovannangeli C. J. Mol. Biol. 2001; 306: 15-24Crossref PubMed Scopus (36) Google Scholar). Corrections for spectrophotometric instability were made by subtracting the absorbance at 540 nm from that at 260 nm. The temperature was decreased from 92 °C to 0 °C and increased again to 92 °C at a rate of 0.2 °C/min. All the cooling and heating profiles presented here were reversible. The melting temperature (Tm in °C) was evaluated from melting curves, either directly or after subtraction of the duplex absorption from that of the triplex. The triplex Tm was estimated within ±0.5 °C accuracy, except when the melting profiles of triplex transition presented a weak hypochromism (±1 °C accuracy in Tm). The standard molar enthalpy and entropy changes ( ΔHa0 and ΔSa0) associated with triplex formation were determined according to a two-state model and a van't Hoff analysis (20Marky L. Breslauer K. Biopolymers. 1987; 26: 1601-1620Crossref PubMed Scopus (1108) Google Scholar). SPR Experiments—SPR measurements were performed on a BIAcore 2000™ (BIAcore AB) using a carboxymethylated dextran-coated sensor chip (CM5), as previously described (21Alberti P. Arimondo P.B. Mergny J.L. Garestier T. Helene C. Sun J.S. Nucleic Acids Res. 2002; 30: 5407-5415Crossref PubMed Scopus (35) Google Scholar). Briefly, a controlled amount of streptavidine (∼5500 resonance units) was immobilized on the chip after activation by N-ethyl-N′-(dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide. The unused activated sites were capped by injecting 100 μl of 1 m ethanolamine. One micromolar solutions of 3′-biotinylated hairpin duplexes (PPT duplex and control duplex 5′-GCTAAAGAGAGAGAGAAATCGTTTTCGATTCTCTCTCTCTTTAGCTTTTTTT-Biotin) were prepared in a buffer purchased from BIA-core (0.01 m HEPES, pH 7.4, 0.15 m NaCl, 3 mm EDTA, and 0.0005% surfactant P20), and duplex injection was controlled to obtain a 1500-resonance unit increase. Serial dilutions of TFO were prepared in 10 mm sodium cacodylate, 10 mm MgCl2, 150 mm NaCl, and 0.0005% surfactant P20 at different pH values (pH 6, 6.5, or 7). These triplexforming buffer conditions are the same as the ones used in UV absorption melting experiments. G-containing TFOs, such as the 15TCG sequence, can self-associated to form G-quadruplex structures, thus decreasing the effective concentration of TFO. To prevent G-quadruplex formation, stock solutions of 15TCG/LNA and 15TCG/po were alkalitreated (with 50 mm NaOH for 15 min at 25 °C and neutralized with 50 mm HCl) before SPR or spectroscopic experiments. Serial TFO injections were performed (100 μl at a flow rate of 10 μl/min) simultaneously on the hairpin duplex containing the target PPT sequence and on the control duplex lacking the PPT sequence. All the sensorgrams were corrected by subtraction of the control duplex signal, reflecting mainly bulk index changes. For thermodynamic analyses, SPR experiments were carried out at four fixed temperatures (20 °C, 25 °C, 30 °C, and 37 °C). The sensorgrams were analyzed using BIAevaluation 3.0 software. The association rate constant kon was determined by the linear fitting of the apparent association rate constant (kapp = kon × [TFO] + koff) at different TFO concentrations ([TFO]), in agreement with a two-state model. The dissociation profile could be fitted by a bi-exponential model. The fast component was neglected because it could arise from a fast relaxation of the sensor chip matrix, as previously discussed (21Alberti P. Arimondo P.B. Mergny J.L. Garestier T. Helene C. Sun J.S. Nucleic Acids Res. 2002; 30: 5407-5415Crossref PubMed Scopus (35) Google Scholar). The mass transport effects were negligible under our experimental conditions. Electrophoresis Mobility Shift Assay—PPT or mutPPT hairpin intramolecular duplexes were 5′-end-labeled with [γ-32P]ATP (3000 Ci/mmol; Amersham Biosciences) by T4 polynucleotide kinase (Promega). The duplex (50 nm) was incubated with increasing concentrations of TFO in a buffer containing 50 mm HEPES, pH 7.2, 150 mm NaCl, 10 mm MgCl2, 0.5 mm spermine, and 10% sucrose (at room temperature overnight). The non-denaturing polyacrylamide gel (15%) was run at 37 °C in a buffer containing 50 mm HEPES, pH 7.2, and 5 mm MgCl2. Gels were scanned with a PhosphorImager, and results were quantified using the ImageQuant software (Amersham Biosciences). The level of complex formation was estimated by the TFO concentration at which 50% of complex was formed (C50). Restriction Enzyme (DraI) Protection Assay—The pCMV(+)PPT/luc plasmid contains the PPT sequence overlapping one of the seven DraI sites. It was used as a substrate for a restriction enzyme protection assay. For the cleavage assay, the pCMV(+)PPT/luc plasmid was incubated at 37 °C for 20 min with increasing amounts of oligonucleotides in 50 mm HEPES, pH 7.2, 50 mm NaCl, 10 mm MgCl2, and 0.5 mm spermine in the presence of DraI enzyme. The fragments generated by DraI cleavage in the absence of TFO are 3654-, 1936-, 1718-, 692-, 683-, 534-, and 19-bp long. A 3654-bp fragment corresponding to the addition of the 1936- and 1718-bp fragments was obtained when TFO-induced inhibition of DraI cleavage occurred. The extent of triplex-mediated inhibition of DraI cleavage was assessed by gel electrophoresis (0.8% agarose gel) and quantitated. The TFO concentration that gave 50% inhibition (IC50) was then evaluated. In Vitro Transcription Assay—Transcription assays were performed using the pSP-F47 plasmid that contains the PPT sequence between T7 and SP6 promoters. The plasmid was linearized by BspEI for T7 transcription and synthesis of purine/PPT-containing RNA (660-nt long) or by Bsu36I for synthesis of pyrimidine/PPT-containing RNA (597-nt long). The linearized plasmids (0.5 μg) were used for in vitro transcription assays in the presence of increasing amounts of TFOs in a buffer containing 40 mm Tris-HCl, pH 7.2; 6 mm MgCl2; 2 mm spermidine; 4 mm dithiothreitol; 1 unit/μl RNase inhibitor in presence of 500 μm ATP, CTP, and UTP; 100 μm GTP; and 0.3 μm [α-32P]GTP. Transcription was initiated by addition of 25 units of phage RNA polymerase (T7 or SP6). After 5 min at 37 °C, transcription reactions were terminated by ethanol precipitation (10 volumes of ethanol was added to samples with 0.3 m sodium acetate and 15 μg of glycogen). The transcription products were analyzed by electrophoresis on 6% polyacrylamide gels, and quantifications (±10%) were obtained by PhosphorImager analysis. The percentages were corrected for transcript length effects, taking into account that the transcripts were uniformly radiolabeled. Cell Cultures and Transient Expression Assay—The P4-CCR5 cells were derived from HeLa cells (22Charneau P. Mirambeau G. Roux P. Paulous S. Buc H. Clavel F. J. Mol. Biol. 1994; 241: 651-662Crossref PubMed Scopus (328) Google Scholar) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. P4-CCR5 cells were used for transfection of oligonucleotides and reporter vectors. Transfections were performed using the cationic activated dendrimer Superfect (Qiagen). Typically, 0.1 μg of pCMV(+/–)-PPT/luc plasmid, 0.1 μg of pTet-On activator plasmid, 0.003125 μg of pRL-CMV plasmid, and various amounts of oligonucleotides were mixed with Superfect (1.5 μl) in a total volume of 12 μl (serum-free medium). The mixture was prepared for triplicates and added to P4-CCR5 cells (13,750 cells/well in a 96-well plate in 88 μl of serum-containing medium in the presence of doxycycline, which was necessary for CMV promoter induction). After cell lysis (in 30 μl of Passive Lysis Buffer; Promega), activities of both luciferases (firefly and Renilla) were measured in the same cell extract using the dual-luciferase assay kit (Promega), and GFP expression was measured as well (15 μl of lysate in 80 μl of phosphate-buffered saline). Luciferase and GFP expressions were measured with a luminometer/fluorometer (Victor™-Wallac). The modulation of firefly luciferase expression by oligonucleotide treatment was quantitated by evaluation of the firefly/Renilla ratio; the specificity of triplex-induced inhibition was evaluated by the GFP/Renilla ratio. Each experiment was repeated at least three times, and values are presented as the mean of a triplicate (±S.D.) from a representative experiment. RNA Analysis—Total cellular RNA was prepared (RNeasy Mini; Qiagen). Firefly and Renilla luciferase RNAs were subjected to competitive reverse transcription-PCR (for details, see Ref. 23Diviacco S. Norio P. Zentilin L. Menzo S. Clementi M. Biamonti G. Riva S. Falaschi A. Giacca M. Gene (Amst.). 1992; 122: 313-320Crossref PubMed Scopus (250) Google Scholar). 2E. Brunet, M. Corgnali, L. Perrouault, U. Asseline, J. Wengel, and C. Giovannangeli, manuscript in preparation. Briefly two sets of two PCR primers were designed for each luciferase RNA; two competitor DNAs (firefly and Renilla) were used and co-amplified with the RNA sample. For each luciferase, the two amplified products (from sample and competitor, which are 153- and 192-bp long for firefly and 250- and 294-bp long for Renilla, respectively) were separated using an 8% PAGE in Tris borate-EDTA and quantitated by PhosphorImager analysis. Tight and Specific Binding of LNA-modified TFOs in the (T,C,G)-Motif—In the present work we have studied the binding and biological properties of a series of LNA-modified TFOs (TFO/LNAs) directed against a 16-bp-long oligopyrimidine· oligopurine sequence (named PPT). All the sequences and their LNA content are shown in Fig. 1. The TFOs (15TCG and 16TC) have been described previously (25Faria M. Wood C.D. Perrouault L. Nelson J.S. Winter A. White M.R. Helene C. Giovannangeli C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3862-3867Crossref PubMed Scopus (132) Google Scholar). Both bind parallel to the purine-containing strand of the duplex by Hoogsteen hydrogen bonding. Because fully modified TFO/LNA failed to hybridize to double-stranded DNA (7Obika S. Uneda T. Sugimoto T. Nanbu D. Minami T. Doi T. Imanishi T. Bioorg. Med. Chem. 2001; 9: 1001-1011Crossref PubMed Scopus (151) Google Scholar), only a few LNA modifications (five to nine) were introduced. For the (T,C,G)-containing sequence, three TFO/LNAs were designed: the 15TCG(1)/LNA is composed of alternating LNA and DNA nucleotides all along the sequence (8 LNA/15 nt); the 15TCG(2)/LNA contains LNA modifications only in the T-rich part of the sequence (5 LNA/15 nt); and the 16TCG/LNA is a modified version of the 15TCG(2)/LNA with two additional LNA modifications at the 3′-end of the sequence to increase resistance to 3′-exonucleases (26Kurreck J. Wyszko E. Gillen C. Erdmann V.A. Nucleic Acids Res. 2002; 30: 1911-1918Crossref PubMed Scopus (452) Google Scholar), and it was used in cellular experiments. The 16TC/LNA sequence is the pyrimidine analogue of the 15TCG(1)/LNA and was designed as a reference to compare the (T,C)- and (T,C,G)-motifs. The thermal stability of the different triplexes formed by these TFO/LNAs and the PPT target sequence was first determined by UV absorption melting experiments at neutral pH. Representative UV melting profiles of the triplexes formed by 15TCG/LNA and 16TC/LNA are shown in Fig. 2; the melting temperature values (Tm) are summarized in Table I. The presence of LNA modifications strongly increases the triplex-forming ability of the oligomers compared with the isosequential phophodiester in the (T,C)-motif (as described previously) (9Torigoe H. Hari Y. Sekiguchi M. Obika S. Imanishi T. J. Biol. Chem. 2001; 276: 2354-2360Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 27Sun B.W. Babu B.R. Sorensen M.D. Zakrzewska K. Wengel J. Sun J.S. Biochemistry. 2004; 43: 4160-4169Crossref PubMed Scopus (66) Google Scholar) and in the (T,C,G)-motif. At neutral pH and physiological concentrations of monovalent cations (150 mm), the highest thermal stability was obtained with the sequences 15TCG(1)/LNA and 15TCG(2)/LNA (Tm = 58 °C and 55 °C, respectively): a 21 °C and 18 °C increase (respectively) in the melting temperature was observed compared with the corresponding pyrimidine sequence 16TC/LNA (Tm = 37 °C). The presence of three additional LNA modifications in the G-rich portion of the 15-TCG sequence slightly enhanced triplex stability (ΔTm(15TCG(1)/LNA–15TCG(2)/LNA) = 3 °C, i.e. 1 °C/(LNA-G nucleotide)). Compared with the isosequential phospodiester 15TCG/po, the presence of LNA modifications resulted in a strong increase in Tm (4.9 °C/LNA nucleotide and 7.2 °C/LNA nucleotide for 15TCG(1)/LNA and 15TCG(2)/LNA, respectively). Two additional LNA modifications at the 3′-end of the 15TCG(2)/LNA sequence did not significantly influence triplex stability as measured by the Tm (ΔTm(16TCG/LNA–15TCG(2)/LNA) = 1 °C). This result is consistent with the hypothesis that the 3′-end nucleotide of the 16TCG/LNA sequence is not involved in base triplet formation as already observed for the 16TCG/po (28Giovannangeli C. Rougee M. Garestier T. Thuong N.T. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8631-8635Crossref PubMed Scopus (137) Google Scholar).Table IMelting temperatures (Tm values) of the different TFOs with the PPT target duplexTFOpHTm Target:PPT duplex°C15TCG/po7.01915TCG(1)/LNA7.05815TCG(2)/LNA7.05516TCG/LNA7.05616TC/LNA7.03716TC/LNA6.54716TC/po5.533 Open table in a new tab In order to assess the specificity of duplex recognition by the 15TCG/LNA sequences, we performed three different assays (Table II). In two of them, we compared the TFO/LNA binding to the PPT duplex target and to the mutPPT duplex containing two mutations (5′-AAAAGAAAAAGGAGGA-3′; mutations are shown in bold). First, using UV absorption melting experiments, we showed that 15TCG(1)/LNA and 15TCG(2)/LNA could discriminate between the wild-type and the mutated target with a large decrease in Tm (ΔTm = 27 °C and 19 °C, respectively). It should be noted that nine contiguous triplets could theoretically be formed with the A4GA4 sequence on both of the targets (PPT and mutPPT). Second, the specificity of triplex formation was confirmed by gel shift experiments. Increasing amounts of the different TFO/LNAs were incubated in the presence of the PPT or mutPPT duplexes at 37 °C and neutral pH (see “Materials and Methods”). TFO concentrations required for 50% of complex formation (C50) were estimated. Under these conditions, the two 15TCG/LNA sequences present affinities for the PPT target in the same range (C50(15TCG(1)/LNA) = 0.4 μm;C50(15TCG(2)/LNA) = 0.15 μm). Under our experimental conditions (150 mm NaCl), the 15TCG sequences might self-associate; a different degree of self-association for 15TCG(1)/LNA and 15TCG(2)/LNA could explain the slight difference in apparent binding affinities. No binding was detected on the mutPPT duplex up to 2.5 μm TFO/LNAs (at this concentration, the triplex was completely formed on PPT duplex), ensuring specificity of DNA target recognition. Third, we determined the capacity of TFO/LNAs to interfere with restriction enzyme cleavage specifically at the triplex site. Indeed, the sequence at the 5′-side of the PPT oligopurine strand provides a recognition site for the restriction enzyme DraI, which overlaps the triplex site on 3 bp (Fig. 1). This enzyme cleaves at the junction of the triple helix site (TTT↓AAA), and triplex formation inhibits DNA cleavage (29Giovannangeli C. Diviacco S. Labrousse V. Gryaznov S. Charneau P. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 79-84Crossref PubMed Scopus (166) Google Scholar). The pCMV(+)-PPT/luc plasmid contains the PPT site and seven DraI sites. In the presence of TFO, DraI cleavage was impaired only on the site overlapping the PPT sequence: inhibition of cleavage at this site led to the appearance of a unique longer 3654-bp fragment and the disappearance of the 1936- and 1718-bp fragments. TFO concentrations required for 50% inhibition (IC50) of DraI cleavage were determined from the concentration dependence of cleavage inhibition for the two 15TCG/LNA TFOs (IC50(15TCG(1)/LNA) ≈ 0.3 μm and IC50(15TCG(2)/LNA) ≈ 0.2 μm). Under the same conditions, the isosequential phosphodiester TFO did not inhibit cleavage at 37 °C up to a concentration of 10 μm. The level of DraI cleavage inhibition specifically at the triplex site induced by TFOs reflects both the specificity and the stability of triplex formation.Table IISpecificity of G-containing TFO/LNA bindingTFO/LNAΔTmaTm values of different G-containing TFO with PPT or mutPPT duplexes (see oligopyrimidine·oligopurine sequences in Fig. 1). Data were obtained in 10 mm cacodylate, 150 mm sodium chloride, and 10 mm magnesium chloride, pH 7. Strand concentrations: 1 μm PPT duplex and 1.5 μm TFO. Estimated error in Tm is ± 0.5 °C (or ± 1 °C for cases indicated by an asterisk, which correspond to a triplex transition presenting a weak hyperchromism). (PPT) target — (mutPPT) targetC50bTFO concentrations for 50% triplex formation estimated from gel shift assays (C50 values). Increasing concentrations of TFO/LNAs were incubated in 150 mm sodium chloride, 50 mm HEPES, pH 7.2, 10 mm magnesium chloride, and 0.5 mm spermine, with either PPT or mutPPT duplex. The samples were analyzed by electrophoresis in 15% non-denaturing polyacrylamide gel (50 mm HEPES, pH 7.2, 5 mm magnesium chloride) at 37 °C. Estimated error, ±0.05 μm.IC50cConcentration for 50% inhibition of DraI cleavage (IC50 values) by TFO/LNAs. Inhibition of DraI cleavage was evaluated on the pCMV(+)PPT/luc plasmid in 50 mm sodium chloride; 50 mm HEPES, pH 7.2, 10 mm magnesium chloride, and 0.5. mm spermine. Analyses were done by electrophoresis on a 0.8% agarose gel and quantification of DraI digestion profiles. Estimated error, ±0.05 μm.Target: (PPT)(mutPPT)°Cμmμmμm15TCG(1)/LNA58 - 31* = 270.40>2.50.315TCG(2)/LNA55 - 36* = 190.15>2.50.2a Tm values of different G-containing TFO with PPT or mutPPT duplexes (see oligopyrimidine·oligopurine sequences in Fig. 1). Data were obtained in 10 mm cacodylate, 150 mm sodium chloride, and 10 mm magnesium chloride, pH 7. Strand concentrations: 1 μm PPT duplex and 1.5 μm TFO. Estimat" @default.
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