Characterization of the DNA triplex formed by d(TGGGTGGGTGGTTGGGTGGG) and a critical R · Y sequence located in the promoter of the murine Ki‐<i>ras</i> proto‐oncogene

Xodo, Luigi E.

doi:10.1016/0014-5793(95)00829-x

Cited by 19 publications

(19 citation statements)

References 35 publications

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“…Our results are in agreement with previously published results on purine-motif triplexes (Xodo 1995;Alunni-Fabbroni et al 1996;Arimondo et al 1998;Raghavan et al 2005). …”

Section: Circular Dichroism Studiessupporting

confidence: 94%

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

Buske

Bauer²,

Mattick³

et al. 2012

Genome Res.

200

182

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Double-stranded DNA is able to form triple-helical structures by accommodating a third nucleotide strand in its major groove. This sequence-specific process offers a potent mechanism for targeting genomic loci of interest that is of great value for biotechnological and gene-therapeutic applications. It is likely that nature has leveraged this addressing system for gene regulation, because computational studies have uncovered an abundance of putative triplex target sites in various genomes, with enrichment particularly in gene promoters. However, to draw a more complete picture of the in vivo role of triplexes, not only the putative targets but also the sequences acting as the third strand and their capability to pair with the predicted target sites need to be studied. Here we present Triplexator, the first computational framework that integrates all aspects of triplex formation, and showcase its potential by discussing research examples for which the different aspects of triplex formation are important. We find that chromatin-associated RNAs have a significantly higher fraction of sequence features able to form triplexes than expected at random, suggesting their involvement in gene regulation. We furthermore identify hundreds of human genes that contain sequence features in their promoter predicted to be able to form a triplex with a target within the same promoter, suggesting the involvement of triplexes in feedback-based gene regulation. With focus on biotechnological applications, we screen mammalian genomes for high-affinity triplex target sites that can be used to target genomic loci specifically and find that triplex formation offers a resolution of~1300 nt.

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“…Our results are in agreement with previously published results on purine-motif triplexes (Xodo 1995;Alunni-Fabbroni et al 1996;Arimondo et al 1998;Raghavan et al 2005). …”

Section: Circular Dichroism Studiessupporting

confidence: 94%

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

Buske

Bauer²,

Mattick³

et al. 2012

Genome Res.

200

182

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“…6 that only with 20AG we observed a complete transformation of the duplex into triplex (see curves at 37°C and 4S"C), while with 20GT about 80% of the duplex is transformed into triplex. This should be due to the finding that the triplex formed by 20AG is thermodynamically stable (ln, > 60°C) at all temperatures considered (20"C, 37"C, 4.5"C), whereas the triplex formed by 20GT (t,,=4S°C) is slightly dissociated at both 37 and 45°C (Xodo, 1995b; AlunniFabbroni et al, 1996b). As the reaction of triplex formation is bimolecular, increasing the TFO concentration should increase the rate of triplex formation.…”

Section: 'Cgagggagggagggaggaagggagggaggg 5'agggagggaggaagggagggagggamentioning

confidence: 97%

“…This means that the second-order constants k , are directly obtained from k,,,,-k, [TFO], (Table 2). This approximation however does not hold for the 20GT triplex, its stability being lower compared to that of the 20AG triplex (t,=45 "C versus t,, > 60°C) (AlunniFabbroni et al, 1996;Xodo, 1995b). With the 20GT triplex the approximation kc,,,,-k, [TFO], is valid only at 20"C, but not at 37 "C and 45 "C, where the dissociation becomes significant.…”

Section: 'Cgagggagggagggaggaagggagggaggg 5'agggagggaggaagggagggagggamentioning

confidence: 99%

A Kinetic Study of Triple‐Helix Formation at a Critical R · Y Sequence of the Murine c‐Ki‐ras Promoter by (A,G)‐ and (G,T) Oligonucleotides

Xodo¹,

Pirulli²,

Quadrifoglio³

1997

European Journal of Biochemistry

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(20AG), d(TGGGTGGGTGGTTGGGTGGG) (20GT) and a 29-bp polypurine-polypyrimidine sequence located in the c-Ki-ms promoter (D) was studied by electrophoretic experiments in 50 mM Tridacetate, pH 7.4, 50 mM NaC1, 5 mM MgC1,. Rates of triplex formation were determined at three different temperatures (20°C, 37 "C and 45 "C), under pseudo-first order conditions obtained by using the triplex-forming oligonucleotide (TFO) 500-fold in excess over the target duplex ( 5 nM). Measurements at TFO/target ratios of 20 and 100 were also carried out. At 37°C the pseudo first-order constants, k,,,,,, were 18.9X10-' s-' for 20AG and 13.0X10-5 s ' for 20GT, yielding association half-lives of 1 h and 1.5 h, respectively.Second-order association constants were found to be in the order of 10' M-' s-': these are slightly lower if compared with those measured for triplex formation by polypyrimidine (C,T) oligonucleotides Mol. Bid. 62,. Dissociation rate constants, k I, were indirectly obtained from equilibrium constants (&) and found to be, at 37"C, 6.7X10-7 s I and 5.4X10-' s-I for 20AG and 20GT, respectively. From the rate constants obtained at 20"C, 37°C and 45°C we estimated activation energies of triplex formation between D plus 20AG and D plus 20GT of respectively 1 3 4 2 2 9 and 88?21 kJ/mol. Moreover, the activation energies for the reaction of triplex dissociation were 385 -C 50 kJ/mol for 20AG and 330 -C-42 kJ/mol for 20GT. Decreasing the TFO/target ratio from 500 to 100 or 20, we observed a concomitant decrease of the association rate, in keeping with the finding that triplex formation occurs through a bimolecular process. We found that the effect of salt on triplex formation is rather complex, as, the addition of 2 mM spermidine boosted the binding rate of 20GT, but slightly reduced that of 20AG; the increase of NaCl from SO mM to 100 mM or 150 mM decreased the rate of triplex formation. Finally, the biological implications of the kinetic behaviour exhibited by the two triplex-forming oligonucleotides specific for the c-Ki-rus promoter are discussed.Keywords: c-Ki-rus promoter; R . R . Y triple helices; kinetics; activation energy.Targeting synthetic triplex-forming oligonucleotides (TFO) against critical promoter sequences is the basis of a new strategy aiming at the selective inhibition of gene expression (Cooney et al

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“…Thus the CD spectrum of parGT recorded in 2 M NaCl, 5 mM ZnCl 2 corresponds to an intramolecular triple helical structure. We notice that the CD signature of this parallel GT triplex presents in the high wavelength part of the spectum two negative bands (266 and 282 nm) which is different from the classical CD signature of antiparallel triplexes which in this region present only one negative band around 278 nm (16,39,40).…”

Section: Discussionmentioning

confidence: 62%

“…We notice that the spectrum recorded at 40°C already looks very much like that of the duplex. Thermal denaturation can be characterized by monitoring ellipticity changes of different CD bands (39,40). Figure 4 (bottom) shows the plot of the ellipticity measured at 266 nm with increasing temperature.…”

Section: Oligonucleotide Structure In the Presence Of Zinc Ionsmentioning

confidence: 99%

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn2+ ions

Khomyakova¹

2000

Nucleic Acids Research

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Characterization of the DNA triplex formed by d(TGGGTGGGTGGTTGGGTGGG) and a critical R · Y sequence located in the promoter of the murine Ki‐ras proto‐oncogene

Cited by 19 publications

References 35 publications

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

Triplexator: Detecting nucleic acid triple helices in genomic and transcriptomic data

A Kinetic Study of Triple‐Helix Formation at a Critical R · Y Sequence of the Murine c‐Ki‐ras Promoter by (A,G)‐ and (G,T) Oligonucleotides

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn2+ ions

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