FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1569085750> ?p ?o ?g. }

• W1569085750 endingPage "7303" @default.
• W1569085750 startingPage "7295" @default.
• W1569085750 abstract "We have targeted the d(G3A4G3)•d(C3T4C3) duplex for triplex formation with d(G3T4G3) in the presence of MgCl2. The resulting triple helix, d(G3T4G3)∗d(G3A4G3)•d(C3T4C3), is considerably weaker than the related triplex, d(G3A4G3)∗d(G3A4G3)•d(C3T4C3), and melts in a biphasic manner, with the third strand dissociating at temperatures about 20−30°C below that of the remaining duplex. This is in distinct contrast to the d(G3A4G3)∗d(G3A4G3)•d(C3T4C3) triplex, which melts in essentially a single transition. Gel electrophoresis under non-denaturing conditions shows the presence of the d(G3T4G3)∗d(G3A4G3)•d(C3T4C3) triplex as a band of low mobility compared to the duplex or the single strand bands. Binding of the d(G3T4G3) third strand and the purine strand of the duplex can be monitored by imino proton NMR spectra. While these spectra are typically very broad for intermolecular triplexes, the line widths can be dramatically narrowed by the addition of two thymines to both ends of the pyrimidine strand. Thermodynamic analysis of UV melting curves shows that this triplex is considerably less stable than related triplexes formed with the same duplex. The orientation of the third strand was addressed by a combination of fluorescence energy transfer and UV melting experiments. Results from these experiments suggest that, in the unlabeled triplex, the preferred orientation of the third strand is parallel to the purine strand of the duplex. We have targeted the d(G3A4G3)•d(C3T4C3) duplex for triplex formation with d(G3T4G3) in the presence of MgCl2. The resulting triple helix, d(G3T4G3)∗d(G3A4G3)•d(C3T4C3), is considerably weaker than the related triplex, d(G3A4G3)∗d(G3A4G3)•d(C3T4C3), and melts in a biphasic manner, with the third strand dissociating at temperatures about 20−30°C below that of the remaining duplex. This is in distinct contrast to the d(G3A4G3)∗d(G3A4G3)•d(C3T4C3) triplex, which melts in essentially a single transition. Gel electrophoresis under non-denaturing conditions shows the presence of the d(G3T4G3)∗d(G3A4G3)•d(C3T4C3) triplex as a band of low mobility compared to the duplex or the single strand bands. Binding of the d(G3T4G3) third strand and the purine strand of the duplex can be monitored by imino proton NMR spectra. While these spectra are typically very broad for intermolecular triplexes, the line widths can be dramatically narrowed by the addition of two thymines to both ends of the pyrimidine strand. Thermodynamic analysis of UV melting curves shows that this triplex is considerably less stable than related triplexes formed with the same duplex. The orientation of the third strand was addressed by a combination of fluorescence energy transfer and UV melting experiments. Results from these experiments suggest that, in the unlabeled triplex, the preferred orientation of the third strand is parallel to the purine strand of the duplex. DNA triple helix formation has been the focus of much attention recently in terms of its potential use as a method for selective regulation of gene expression(1Helene C. Anticancer Drug Des. 1991; 6: 569-584PubMed Google Scholar). Oligonucleotides can inhibit protein synthesis in several different ways. In the antisense strategy, an oligonucleotide binds to a targeted mRNA molecule in a sequence-specific manner to prevent subsequent translation of the message into protein. Alternatively, binding of an oligonucleotide directly to a gene or gene promoter, via triplex formation, can arrest or block transcription. The process of down-regulating gene expression through triple helix formation is referred to as the antigene strategy. Triple helix formation has also been utilized in the development of artificial nucleases, created by tethering a cleaving agent to a triplex-forming oligonucleotide(2Strobel S.A. Dervan P.B. Science. 1990; 249: 73-75Crossref PubMed Scopus (182) Google Scholar, 3Strobel S.A. Doucette-Stamm L.A. Riba L. Housman D.E. Dervan P.B. Science. 1991; 254: 1639-1642Crossref PubMed Scopus (188) Google Scholar, 4Strobel S.A. Dervan P.B. Nature. 1991; 350: 172-174Crossref PubMed Scopus (129) Google Scholar). This leads to breaks in double-stranded DNA at very specific sites. Several studies have demonstrated the feasibility of using oligonucleotides in gene regulation(5Duval-Valentin G. Thuong N.T. Helene C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 504-508Crossref PubMed Scopus (257) Google Scholar, 6Ing N.H. Beekman J.M. Kessler D.J. Murphy M. Jayaraman K. Zendegui J.G. Hogan M.E. O'Malley B.W. Tsai M.J. Nucleic Acids Res. 1993; 21: 2789-2796Crossref PubMed Scopus (103) Google Scholar, 7Roy C. Nucleic Acids Res. 1993; 21: 2845-2852Crossref PubMed Scopus (47) Google Scholar). Several properties need to be better understood in order to design optimally effective therapeutic agents based on triplex formation. These include sequence specificity of triplex formation, stability of the complex formed, delivery of the oligonucleotide into cells, and resistance of the oligonucleotides to endogenous nucleases. The primary requirement for triple helix formation is a homopurine-homopyrimidine sequence in the target duplex. Typically, the third strand sequence is also homopurine or homopyrimidine. Depending on the nature of the third strand, there are two main categories of triple helices: pyrimidine∗ purine∗pyrimidine (pyr∗pur•pyr) or purine∗purine•pyrimidine (pur∗pur•pyr), where ∗ represents Hoogsteen base pair formation between the third strand and the purine strand of the duplex and • denotes the Watson-Crick base pair of the duplex. Most studies to date have focused on pyr∗pur•pyr triplexes. This type of triplex is more stable at low pH because the cytosines on the third strand require protonation in order to form Hoogsteen hydrogen bonds with the purine strand of the duplex. In contrast, pur∗pur•pyr triplexes are stable at neutral pH. Thermal denaturation studies on pur∗pur•pyr triplexes often reveal a single transition, suggesting the simultaneous dissociation of all three strands(8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar). In most examples of pyr∗pur•pyr triplexes, a biphasic transition is observed, with the third strand dissociating at a lower temperature than the duplex(9Pilch D.S. Brousseau R. Shafer R.H. Nucleic Acids Res. 1990; 18: 5743-5750Crossref PubMed Scopus (116) Google Scholar, 10Plum G.E. Park Y.W. Singleton S.F. Dervan P.B. Breslauer K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9436-9440Crossref PubMed Scopus (242) Google Scholar, 11Scaria P.V. Shafer R.H. J. Biol. Chem. 1991; 266: 5417-5423Abstract Full Text PDF PubMed Google Scholar, 12Mergny J.L. Duval-Valentin G. Nguyen C.H. Perrouault L. Faucon B. Rougee M. Montenaygarestier T. Bisagni E. Helene C. Science. 1992; 256: 1681-1684Crossref PubMed Scopus (313) Google Scholar). This suggests that a pyrimidine third strand may be less stable, in general, than a purine third strand. Unlike the Watson-Crick base-paired double helix, in which the two strands are bound antiparallel to each other, the third strand of a triple helix can be either parallel or antiparallel with respect to the purine strand to which it is hydrogen-bonded. The polarity of the third strand in the triplex is dependent on the sequence as well as the base composition(13Sun J.S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 14Thuong N.T. Helene C. Angew. Chem. Int. Ed. Engl. 1993; 32: 666-690Crossref Scopus (712) Google Scholar). A homopyrimidine third strand containing cytosine and/or thymine binds to the purine strand in a parallel orientation. The binding of a homopurine oligonucleotide to the purine strand depends on the sequence. Experimental evidence has been reported for both parallel (15Ouali M. Letellier R. Sun J.S. Akhebat A. Adnet F. Liquier J. Taillandier E. J. Am. Chem. Soc. 1993; 115: 4264-4270Crossref Scopus (46) Google Scholar) and antiparallel (16Chen F.M. Biochemistry. 1991; 30: 4472-4479Crossref PubMed Scopus (80) Google Scholar) orientations of the third strand in triplexes composed solely of G∗G•C triplets. But if the third strand contains both Gs and As, the polarity of the third strand will be antiparallel with respect to the purine strand(8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar, 17Beal P.A. Dervan P.B. Science. 1991; 251: 1360-1363Crossref PubMed Scopus (576) Google Scholar), since As with anti-glycosidic bond conformations can form only reverse-Hoogsteen hydrogen bonds (13Sun J.S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar) and hence direct the oligonucleotide in an antiparallel orientation. Hogan and co-workers (18Orson F.M. Thomas D.W. McShan W.M. Kessler D.J. Hogan M.E. Nucleic Acids Res. 1991; 19: 3435-3441Crossref PubMed Scopus (187) Google Scholar, 19McShan W.M. Rossen R.D. Laughter A.H. Trial J. Kessler D.J. Zendegui J.G. Hogan M.E. Orson F.M. J. Biol. Chem. 1992; 267: 5712-5721Abstract Full Text PDF PubMed Google Scholar) have reported antigene activity of oligonucleotides composed of G and T bases. However, there have been only a limited number of physical studies on triplexes formed by such sequences. Helene and co-workers (13Sun J.S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar, 20Sun J.S. De Bizemont T. Duval-Valentin G. Montenay-Garestier T. Helene C. C. R. Acad. Sci. III. 1991; 313: 585-590PubMed Google Scholar) have demonstrated that the orientation of a G, T third strand depends on its sequence. Calculations indicate that, for a 10-mer triplex whose third strand is composed of equal number of G and T bases (with corresponding G and A bases in the purine strand of the target duplex), the third strand will be antiparallel to the duplex purine strand if there are three or more GpT or TpG steps; otherwise it will be parallel. According to the calculations of Sun and Helene(13Sun J.S. Helene C. Curr. Opin. Struct. Biol. 1993; 3: 345-356Crossref Scopus (170) Google Scholar), this can be explained in terms of a balance between the preference for the parallel orientation (Hoogsteen hydrogen bonding) in an all G third strand and the better tolerance for the lack of isomorphism between G∗G•C and T∗A•T triplets in the antiparallel orientation (reverse Hoogsteen hydrogen bonding). Radhakrishnan et al.(21Radhakrishnan I. de los Santos C. Patel D.J. J. Mol. Biol. 1991; 221: 1403-1418PubMed Google Scholar) carried out NMR studies on an intramolecular triplex composed of a third strand containing Gs and one T. These studies provided clear evidence for the distortion induced by insertion of a T∗A•T triplet within a stack of G∗G•C triplets. In earlier studies, we have targeted the d(G3A4G3)• d(C3T4C3) for triple helix formation with either d(C3T4C3), to make a pyr∗pur•pyr triplex(9Pilch D.S. Brousseau R. Shafer R.H. Nucleic Acids Res. 1990; 18: 5743-5750Crossref PubMed Scopus (116) Google Scholar), or with d(G3A4G3), to make a pur∗pur•pyr triplex(8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar). We have recently described the Fourier transform infrared spectrum of the pyr/pur∗pur•pyr intermolecular triplex d(G3T4G3)∗d(G3A4G3)•d(C3T4C3) (22Dagneau C. Liquier J. Scaria P.V. Shafer R.H. Taillandier E. Sarma R.H. Sarma M.H. Proceedings of the Eighth Conversation, State University of New York, Albany NY. Adenine Press, Schenectady, NY1994: 103-111Google Scholar). Here we employ UV, CD, gel electrophoresis, and NMR to further characterize this triplex. While results from fluorescence energy transfer experiments are inconclusive regarding the orientation of the third strand relative to the purine strand of the duplex, UV melting analysis of fluorescently labeled triplexes suggests that the preferred orientation of the third strand is parallel to the purine strand of the duplex in the unlabeled triplex. Oligodeoxynucleotides were synthesized using standard phosphoramidite chemistry on an automated DNA synthesizer as described earlier(8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar). The deprotected oligonucleotides were extensively dialyzed against 1 mM Tris-HCl with several changes of buffer over a period of 2-3 days. The purity of the resulting oligonucleotides was checked by NMR and gel electrophoresis and found to be greater than 95%. Molar extinction coefficients of various oligonucleotides in 1 mM Tris-HCl were determined by phosphate analysis(23Griswold B.L. Humoller F.L. MacIntyre A.R. Anal. Chem. 1951; 23: 192-194Crossref Scopus (64) Google Scholar), and the concentration of the stock solutions was determined using the following values: d(G3A4G3): ∊255 = 11500 cm−1(mol of base)−1L; d(C3T4C3): ∊271 = 8300 cm−1(mol of base)−1L; d(G3T4G3): ∊256 = 9900 cm−1(mol of base)−1L; d(T2C3T4C3T2): ∊269 = 8500 cm−1(mol of base)−1L. All experiments were carried out in buffer containing 10 mM Tris-HCl with 50 mM MgCl2 at pH 7.4. Duplex and triplex samples were prepared by mixing the oligonucleotides at appropriate ratios in the desired buffer, followed by heating at 80°C for about 5 min and cooling slowly to room temperature. The samples were equilibrated at 5°C overnight before use. Labeled oligonucleotides were synthesized using phosphoramidite chemistry on ABI 394 DNA synthesizers. The 3′-labels were coupled to oligonucleotides prepared using the 3′-DMT-C6 amine-ONTM controlled pore glass (Clontech). The 5′-carboxytetramethlyrhodamine was added to a 5′-aminohexyl-derivatized oligonucleotide (Glen Research). All amino oligonucleotides were ion-exchanged to their lithium salts by precipitation from ethanol-acetone as described previously(24Levenson C. Chang C. Innis M.A. Sinsky D.H. White T.J. PCR Protocols: a Guide to Methods and Applications. Academic Press, San Diego1990: 99-112Google Scholar). The crude, derivatized oligonucleotides were dissolved in sodium carbonate buffer (0.25 ml, 0.1 M, pH 9.0) and treated with 5-carboxytetramethylrhodamine-N-hydroxysuccinimidyl ester (Applied Biosystems, 3 μl in Me2SO) or Malachite Green isothiocyanate (1 mg in 50 μl of dry N,N-dimethylformamide, Molecular Probes). After coupling overnight at room temperature, the oligonucleotides were again precipitated as their lithium salts from ethanol-acetone. The oligonucleotides were resuspended in triethylammonium acetate buffer (0.1 M, pH 7.0, buffer A), filtered, and purified by reverse-phase HPLC 1The abbreviation used is: HPLChigh performance liquid chromatography. on a Waters 996 Diode Array Detector system with a Hamilton PRP-1 column (300 × 7 mm) using a gradient of 0-40% acetonitrile in buffer A at a flow rate of 2 ml/min. high performance liquid chromatography. The 5′-Malachite Green oligonucleotides were made directly on the DNA synthesizer using a leuco-Malachite Green phosphoramidite, details of which will be published elsewhere. 2S. G. Will and D. Knowles, manuscript in preparation. The oligonucleotides were coupled to the Malachite Green amidite using normal activation and oxidation steps. On removal from the instrument, the synthesis columns were treated with a solution of freshly prepared iodosobenzene in dichloromethane (0.01 M, 30 s) and washed with more solvent and air-dried. The derivatized controlled pure glass was treated with ammonia for 4 h at 55°C, then the supernatants were passed through NAP-10 columns (Pharmacia LKB Biotechnol), and the desired oligonucleotides were purified by reverse phase-HPLC as described above. The UV absorbance and melting studies were carried out on a Gilford 2600 UV/Vis spectrophotometer equipped with a Gilford 2527 thermoprogrammer or on a Cary 3 spectrophotometer. UV melts were done with a heating rate of 0.25 or 0.3 deg/min. CD spectra were recorded on a Jasco J500A spectropolarimeter using cells of 0.1-, 1.0-, or 10-mm optical pathlength, with temperature controlled by an external circulating water bath. CD spectra reported are the average of eight scans. CD melting experiments were done by manually changing the temperature of the bath and letting it equilibrate at the desired temperature for 5 min. The cell temperature was monitored by attachment of a microprobe directly onto the sample cell. The ellipticity data were collected at various wavelengths, and each point on the melting curve represents the average of 100 readings taken over a period of 100 s. Mixing curves were constructed from data obtained from the CD spectra of samples containing varying mole ratios of duplex and the third strand, with the total concentration of the duplex plus the third strand held constant. Thermodynamic parameters for the formation of the triplex were estimated from the concentration dependence of the thermal melting temperature of the triplex in the concentration range 10 to 850 μM. Since the two transitions were well separated from each other over a wide concentration range, we were able to follow the concentration dependence of each transition separately. Assuming a two-state model for each transition, we analyzed the biphasic melting curves according to the procedure described by Pilch et al.(9Pilch D.S. Brousseau R. Shafer R.H. Nucleic Acids Res. 1990; 18: 5743-5750Crossref PubMed Scopus (116) Google Scholar) using the equation, 1/Tmax = (R/ΔH°)lnC + (ΔS° - 0.188R)/ΔH° for each individual transition. ΔH° and ΔS° are the enthalpy and entropy changes, respectively, associated with the respective structural transitions, Tmax is the temperature corresponding to the maximum of the dA/d(1/T) plot derived from the melting curves, where A is the absorbance, C is the total concentration of the d(G3T4G3) strand (which is equal to the total concentration of each of the other two strands), and R is the gas constant. ΔH° and ΔS° were calculated from the slope and intercept of the straight lines obtained by fitting the data points on 1/Tmaxversus lnC plots by linear regression. The UV melting curves used for the thermodynamic analysis were obtained at a heating rate of 0.25°/min; a window of ± 2°C was used for calculating the derivative. The reversibility of the transitions was checked by heating the sample at 1°C/min up to 90°C followed by cooling at the same rate. No hysterisis was observed in the melting curves showing that the equilibrium is maintained at each point on the melting curve. Gel electrophoresis was carried out under non-denaturing conditions in 15% polyacrylamide gels containing acrylamide and bis(acrylamide) in a 29:1 ratio, cast in 90 mM Tris-borate and 50 mM MgCl2. Samples for electrophoresis were prepared in 90 mM Tris-borate and 50 mM MgCl2 and were diluted into loading buffer containing 90 mM Tris-borate, 50 mM MgCl2, and 5% Ficoll, so that the final concentration ranged from 50 to 60 μM in single strand, duplex, or triplex. Then, 40 μl of each sample were loaded into the wells, and electrophoresis was carried out in buffer containing 90 mM Tris-borate and 50 mM MgCl2, in the cold room (4°C) or at room temperature (25°C) at about 7 V/cm for 24 h. Gels were then visualized by UV shadowing on a fluorescent background and photographed. Proton NMR spectra of the samples were recorded at 500 MHz on a General Electric spectrometer (GN-500) equipped with an Oxford Instruments magnet and a Nicolet 1280 computer. Spectra of the samples were taken in aqueous solutions containing 10 mM Tris-HCl, 0-50 mM MgCl2, and 85% H2O, 15% D2O, at 5 or 20°C. MgCl2 titrations were carried out by adding aliquots of MgCl2 into samples containing the duplex and the third strand in equimolar ratios (1 mM each) dissolved in the buffer. After each addition of MgCl2, the samples were heated at 80°C for 5 min and cooled. The spectra were acquired using a T331 pulse sequence for solvent suppression, with a pulse repetition time of 5 s and interval delay of 109 μs. We have shown previously that the oligonucleotide d(G3T4G3), used here as the third strand for triplex formation, can, in the presence of monovalent cations such as Na+ or K+, self-associate to form a quadruplex structure(25Scaria P.V. Shire S.J. Shafer R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10336-10340Crossref PubMed Scopus (109) Google Scholar). This quadruplex structure can be eliminated by dialysis against 1 mM Tris-HCl buffer. Tris, a bulky cation, does not promote quadruplex formation of d(G3T4G3), nor do the concentrations of Mg+2 used in this study to stabilize the triplex (data not shown). A 1:1 mixture of d(G3T4G3) and the duplex, at typical UV concentrations, shows a melting profile similar to that of the duplex and to that of the d(G3A4G3)∗d(G3A4G3)•d(C3T4C3) triplex, composed of a single transition occurring at a temperature corresponding to the melting temperature of the duplex. However, melting curves for d(G3T4G3)∗d(G3A4G3)•d(C3T4C3) measured at higher concentrations reveal the presence of a broad transition at lower temperature, well below the duplex melting temperature (see below). This low temperature transition represents the dissociation of the third strand from d(G3T4G3)∗d(G3A4G3)•(C3T4C3) and was investigated by additional techniques, such as CD and gel electrophoresis, in order to further characterize this change in conformation. As mentioned above, UV melting curves showed evidence of a low temperature transition only at DNA concentrations higher than those typically used in spectrophotometric studies. Hence, we initiated CD studies at concentrations ranging from 0.1 to 0.5 mM in either single strand, duplex, or triplex. Fig. 1 shows the CD spectra of d(G3T4G3) alone, the d(G3A4G3)•d(C3T4C3) duplex, the duplex and the third strand in a 1:1 ratio, all at the same concentration and in buffer containing 10 mM Tris-HCl and 50 mM MgCl2, along with the mathematical sum of the spectra of the duplex and d(G3T4G3). It is apparent that the CD spectrum of the mixture of duplex and d(G3T4G3) is very different compared to that of the sum of the duplex and the third strand spectra, especially in the long and short wavelength regions. The spectrum of the mixture has three large negative bands centered at 277, 240, and 210 nm, and a positive band around 260 nm. The mathematical sum of the spectra of the duplex and the third strand has no negative band in the 277 and 210 nm regions. The large differences observed in these two spectra clearly indicate interaction between the duplex and the third strand to form the triplex and hence the CD spectrum of the mixture represents the spectrum of the triplex (see below). Thermal denaturation of the d(G3T4G3)∗d(G3A4G3)• d(C3T4C3) triplex was studied by temperature induced changes in the CD spectrum at the same high oligonucleotide concentrations. Fig. 2a shows a typical thermal denaturation profile of the triplex monitored by CD changes at 240 and 277 nm. As the temperature increases, the ellipticity values at the two wavelengths also increase, except for a small decrease at high temperature for the 277 nm band. The change in ellipticity with temperature is biphasic and cooperative for both wavelengths, with the two transitions centered around 38 and 60°C. It is evident from Fig. 2a that the low temperature transition is most readily followed at 277 nm while the high temperature transition is best monitored at 240 nm. Fig. 2b shows the CD melting profile of the duplex at the same two wavelengths. The duplex shows only one transition around 60°C. The ellipticity at 277 nm does not show any cooperative change in the lower temperature region, where the triplex shows a large change. As in the case of the triplex, however, the temperature profile shows a relatively small but cooperative decrease in ellipticity around 60°C. The 240-nm melting curve is also monophasic, with a transition at the same temperature. Hence the transition that occurs around 38°C for the triplex is due to the dissociation of the third strand from the triplex while that at 60°C is due to the dissociation of the duplex into single strands. The differences in the CD spectra elicited by the binding of d(G3T4G3) to the duplex were utilized to construct a mixing curve that provides further evidence for complex formation between the duplex and the third strand and also determines the stoichiometry of the complex formed. Fig. 3 presents the mixing curve for the binding of d(G3T4G3) to the duplex monitored by the CD changes associated with complex formation for the 277 nm band. The titration of the third strand into the duplex was carried out by varying the mole ratio of the duplex and the third strand, keeping the total concentration, duplex plus third strand, constant. The discontinuous change in slope in the mixing curve observed when the mixture contains equal amounts of the duplex and the third strand demonstrates that the stoichiometry of the complex formed is 1 duplex/1 strand of d(G3T4G3). UV melting studies of the triplex showed a marked dependence of the melting temperature on the concentration of the triplex. The concentration dependence of the biphasic melting curves was thus analyzed in order to estimate the thermodynamic parameters for these structural transitions. Fig. 4 shows the dependence of the melting temperature on oligonucleotide concentration, plotted in terms of 1/Tmaxversus lnC for both transitions. The first transition, the dissociation of the d(G3T4G3) strand from the underlying duplex, is more sensitive to the concentration than the second transition, which corresponds to the dissociation of the duplex. The following thermodynamic parameters, relating to formation of the triplex, were obtained from the slope and intercept of the plots in Fig. 4: ΔH° = −19.8 ± 2 kcal/mol and ΔS° = −47 ± 5 cal/mol-deg. The low value of ΔH° is reflected in the large slope observed for the 1/Tmaxversus lnC curve for the first transition. The enthalpy change obtained for triplex formation is less than that reported for triplexes formed by the binding of an all purine or all pyrimidine third strand to the same underlying duplex (8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar, 9Pilch D.S. Brousseau R. Shafer R.H. Nucleic Acids Res. 1990; 18: 5743-5750Crossref PubMed Scopus (116) Google Scholar) and presumably reflects the presence of a mixed purine/pyrimdine third strand. The thermodynamic parameters for dissociation of the underlying duplex were similar to those reported earlier(8Pilch D.S. Levenson C. Shafer R.H. Biochemistry. 1991; 30: 6081-6088Crossref PubMed Scopus (193) Google Scholar, 9Pilch D.S. Brousseau R. Shafer R.H. Nucleic Acids Res. 1990; 18: 5743-5750Crossref PubMed Scopus (116) Google Scholar). Fig. 5 shows the polyacrylamide gel electrophoresis pattern of d(G3A4G3), d(G3T4G3), d(C3T4C3), duplex, and 1:1 mixture of the duplex and d(G3T4G3), all under triplex forming conditions at 4°C (Fig. 5a) and 25°C (Fig. 5b). Lanes 1-3 contain the oligonucleotides d(G3A4G3), d(C3T4C3), and d(G3T4G3), respectively, which run as single bands. Lane 4, containing d(G3A4G3) and d(C3T4C3) in equimolar amounts, again shows a single band due to formation of the d(G3A4G3)•d(C3T4C3) duplex, which possesses a mobility similar to that of the single-stranded oligonucleotides d(G3A4G3) and d(G3T4G3). The single strand d(C3T4C3) migrates faster than the other two oligonucleotides. Lane 5 shows the migration of the sample containing a mixture of all the three oligonucleotides in 1:1:1 ratio. At 4°C, this lane shows a new band that migrates significantly more slowly than the duplex or any of the three oligonucleotides. This new band corresponds to the triplex, which, as one would expect, migrates more slowly than any of the other species. Under the same buffer and salt conditions but at 25°C, this band has disappeared, indicating that the triplex is unstable at this temperature. The single band observed in lane 5 at 25°C is slightly broadened due to comigration of the dissociated third strand with the duplex. This experiment clearly demonstrates the dissociation of the third strand from the triplex at a temperature considerably lower than the duplex dissociation temperature, in accord with the UV and CD melting profiles. We have employed one-dimensional imino proton NMR spectra as a direct probe for monitoring base triplet formation. Fig. 6 shows the exchangeable proton region of the NMR spectrum of the triplex and that of the mixture of the non-interacting duplex and third strand. The imino proton region in the absence of MgCl2 gives a spectrum identical to that of the duplex alone (data not shown). Addition of Mg2+ to the mixture results in the appearance of new resonances accompanied by substantial broadening of all signals. These new peaks arise from hydrogen bonding interactions between the third strand and the purine strand of the duplex. At 20°C, the temperature at which the spectra in the Fig. 6 were recorded, CD melting studies suggest that the triplex may not be completely formed. Lowering the temperature resulted in considerable line broadening, rendering the spectrum essentially unusable for further analysis. In an effort to sharpen the NMR lines, we designed another triplex in which the pyrimidine strand has two extra thymines at each end, the other two strands being same as in the original triplex. With this “overhang” triplex, d(G3T4G3)∗d(G3A4G3)• d(TTC3T4C3TT), we were able to reduce the temperature and maintain reasonably narrow line widths for the imino proton peaks. Fig. 7 provides a comparison of the spectra obtained for this triplex with that of the “blunt end” triplex, d(G3T4G3)∗ d(G3A4G3)•d(C3T4C3), at 5°C. The blunt end spectrum is very broad and featureless whereas the overhang spectrum has fairly well resolved peaks. In this case, the addition" @default.
• W1569085750 created "2016-06-24" @default.
• W1569085750 creator A5017442356 @default.
• W1569085750 creator A5024751077 @default.
• W1569085750 creator A5032510435 @default.
• W1569085750 creator A5034975411 @default.
• W1569085750 date "1995-03-01" @default.
• W1569085750 modified "2023-10-14" @default.
• W1569085750 title "Physicochemical Studies of the d(G3T4G3)∗d(G3A4G3)•d(C3T4C3) Triple Helix" @default.
• W1569085750 cites W1499463091 @default.
• W1569085750 cites W1575281218 @default.
• W1569085750 cites W1769395946 @default.
• W1569085750 cites W1966342019 @default.
• W1569085750 cites W1975051960 @default.
• W1569085750 cites W1985108256 @default.
• W1569085750 cites W1987743680 @default.
• W1569085750 cites W1996058312 @default.
• W1569085750 cites W1998264445 @default.
• W1569085750 cites W1998269261 @default.
• W1569085750 cites W2004059420 @default.
• W1569085750 cites W2012488414 @default.
• W1569085750 cites W2014299588 @default.
• W1569085750 cites W2014806360 @default.
• W1569085750 cites W2023697509 @default.
• W1569085750 cites W2033059897 @default.
• W1569085750 cites W2041265794 @default.
• W1569085750 cites W2043558250 @default.
• W1569085750 cites W2045366550 @default.
• W1569085750 cites W2057476671 @default.
• W1569085750 cites W2058358001 @default.
• W1569085750 cites W2061419481 @default.
• W1569085750 cites W2064245630 @default.
• W1569085750 cites W2065459148 @default.
• W1569085750 cites W2076746045 @default.
• W1569085750 cites W2087155680 @default.
• W1569085750 cites W2089163172 @default.
• W1569085750 cites W2094412953 @default.
• W1569085750 cites W2094588748 @default.
• W1569085750 cites W2096618772 @default.
• W1569085750 cites W2106858736 @default.
• W1569085750 cites W2143626908 @default.
• W1569085750 cites W224616256 @default.
• W1569085750 cites W4251663802 @default.
• W1569085750 doi "https://doi.org/10.1074/jbc.270.13.7295" @default.
• W1569085750 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7706270" @default.
• W1569085750 hasPublicationYear "1995" @default.
• W1569085750 type Work @default.
• W1569085750 sameAs 1569085750 @default.
• W1569085750 citedByCount "37" @default.
• W1569085750 countsByYear W15690857502015 @default.
• W1569085750 countsByYear W15690857502017 @default.
• W1569085750 countsByYear W15690857502022 @default.
• W1569085750 countsByYear W15690857502023 @default.
• W1569085750 crossrefType "journal-article" @default.
• W1569085750 hasAuthorship W1569085750A5017442356 @default.
• W1569085750 hasAuthorship W1569085750A5024751077 @default.
• W1569085750 hasAuthorship W1569085750A5032510435 @default.
• W1569085750 hasAuthorship W1569085750A5034975411 @default.
• W1569085750 hasBestOaLocation W15690857501 @default.
• W1569085750 hasConcept C10919887 @default.
• W1569085750 hasConcept C185592680 @default.
• W1569085750 hasConcept C71240020 @default.
• W1569085750 hasConceptScore W1569085750C10919887 @default.
• W1569085750 hasConceptScore W1569085750C185592680 @default.
• W1569085750 hasConceptScore W1569085750C71240020 @default.
• W1569085750 hasIssue "13" @default.
• W1569085750 hasLocation W15690857501 @default.
• W1569085750 hasOpenAccess W1569085750 @default.
• W1569085750 hasPrimaryLocation W15690857501 @default.
• W1569085750 hasRelatedWork W1531601525 @default.
• W1569085750 hasRelatedWork W1990781990 @default.
• W1569085750 hasRelatedWork W2319480705 @default.
• W1569085750 hasRelatedWork W2384464875 @default.
• W1569085750 hasRelatedWork W2606230654 @default.
• W1569085750 hasRelatedWork W2607424097 @default.
• W1569085750 hasRelatedWork W2748952813 @default.
• W1569085750 hasRelatedWork W2899084033 @default.
• W1569085750 hasRelatedWork W2948807893 @default.
• W1569085750 hasRelatedWork W2778153218 @default.
• W1569085750 hasVolume "270" @default.
• W1569085750 isParatext "false" @default.
• W1569085750 isRetracted "false" @default.
• W1569085750 magId "1569085750" @default.
• W1569085750 workType "article" @default.