Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy Peter Mojzes , Laurent Chinsky , Pierre-Yves Turpin & Pavol Miskovsky To cite this article: Peter Mojzes , Laurent Chinsky , Pierre-Yves Turpin & Pavol Miskovsky (1992) Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy, Journal of Biomolecular Structure and Dynamics, 10:1, 181-194, DOI: 10.1080/07391102.1992.10508637 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10508637
Published online: 21 May 2012.
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Date: 15 November 2015, At: 06:06
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 10, Issue Number 1 (1992), "'Adenine Press (1992).
Downloaded by [New York University] at 06:06 15 November 2015
Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy Peter Mojzes2 Laurent Chinsky1, Pierre-Yves Turpini,• and Pavol Miskovsky3 1
L.P.C.B. (CNRS UA 198) Institut Curie and Universite P. et M. Curie 75231 Paris Cedex 05, France 2
Institute of Physics Charles University CS-12116 Prague 2, Czechoslovakia 3
Department of Biophysics Safarik University CS-04054 Kosice, Czechoslovakia Abstract The conformational changes of poly( d2NH2A-dT) in aqueous solution, induced by increasing the NaCI concentration from O.IM to 4M, have been monitored by ultraviolet resonance Raman spectroscopy, in using the 222-, 257- and 281 nm excitation wavelengths. These changes have been interpreted in comparing the polymer spectra to those of the mononucleotide compounds on one hand, and to those of other alternating purine-pyrimidine polymers on the other hand, i.e. poly(dG-dC) and poly( dA-dT) which showed a B to Z transition in going from low- to high salt concentrations. The high salt poly( d2NH2A-dT) spectra do not show any Raman marker line of the Z conformation. The spectroscopic results indicate that most ofthe ribose puckering goes from C2' -endo/anti to C3' -endo/anti in increasing the salt concentration. In addition the base stacking interactions, to which the resonance Raman effect is very sensitive, are not drastically changed upon salt variations. Thus the high salt structure of poly( d2NH 2A-dT) remains a right-handed helix, likely under a dominant A conformation.
Introduction The salt-induced conformational transition of a self-complementary hexanucleotide consisting of deoxythymidine (d1) and 2-aminodeoxyadenosine (d2NH:zA), i.e. (dT-d2NH2A)3, has been at first monitored by CD spectroscopy ( 1). The authors proposed that it was similar to a B-Z transition (rigth to left-handed helix), on the • Author to whom correspondence should be addressed.
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basis of similarities with the B-Z transition of poly(dG-dC), i.e. essentially the sign inversion of the first CD extremum which is usually correlated to the inversion of helix handedness. The same cooperative transition, induced by a gradual increase ofNaCl concentration, has been observed in aqueous solutions of(d2NH2A-dT)n oligomers (2) and poly(d2NH2A-dT) alternating copolymers (3}, and monitored by CD, IR, UV-visible and 31 P NMR spectroscopies. The CD results could again be interpreted in terms ofB-Z transitions. Moreover, this was supported by the resemblances between the 2NH~ and guanine molecules, due to the 2-amino substitution in adenine : as in the case of in poly(dG-dC), this bulky amino group in the minor groove of poly( d2NH~-dT) , disrupting a regular spine of hydration, was supposed to be the crucial factor destabilizing the rigth-handed B-ONA. thus allowing a transition to the left-handed Z form (4). This was correlated to the sign inversion of poly(d2NH2A-dT) high-salt CD spectra (3), in opposition to what is observed in poly(dA-dT} CD spectra under the same salt conditions. However Howard et al. (2) showed thatthe 31 P NMR spectrum oflow-salt (d2NH2AdT}n consisted in two resonances of approximately equal intensity, separated by 32 Hz. The same doublet (separation of24 Hz) had been observed for low-salt poly(dAdT} (5) and assigned to an alternating conformation (alternating B-ONA) of the phosphodiester backbone of this polymer in solution. On increasing the salt concentration in poly(d2NH~-dT) solution, the separation of these two resonances increased up to 71 Hz, along with a downfield shift (2). This suggests further differentiation between two distinct phosphorus environments in the phosphodiester backbone, probably in an alternating array. The changes bear a cooperative character, suggesting a transition between two distinct alternating conformations. In addition, the interpretation based on CD measurements has been questioned by 20 NOE proton NMR results (6,7), which established that the conformation ofboth d2NH2A and dT residues in the high-salt form ofpoly(d2NH2A-dT) is C3'-endo/ anti, whereas the purine nucleosides in Z-ONA should be C3'-endo/syn. Consequently, Borah et al. concluded thatthe polynucleotide conformation would neither be the Z nor the B form, but instead an A form or closely related structure (6) having pyrimidine as well as purine residues in the C3'-endo/anti conformation. On the other hand, preliminary X-ray diffraction results on poly( d2NH~-dT) salt fibers (at 98% r.h.) have been recently published (8). In terms of overall distribution of intensities, the diffraction patterns suggest a structure which is close to that of ADNA. although the helical periodicity (12 base pairs per tum) and the crystal packing appreciably differ from those of A-DNA. and from those of other known deoxyribonucleodide structures as well. Moreover, the handedness ofthe poly(d2NH2AdT) helix in salt fibers was not established, thus the question whether the poly(d2NH~ dT) undergoes inversion of its handedness on salt-induced transition still remains open. In recent CD studies, Vorlickova et al. have shown that the conformation induced by various alcohols (9) or by sub millimolar concentrations ofMg 2+ cations (10) in low-salt poly(d2NH2A-dT) aqueous solutions is similar to that of the high-salt solution
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of the same polymer. They also observed strong similarities between the high-salt form of poly(d2NH0--dT) and what they had observed in increasing drastically the CsF concentration in a solution of poly(dA-dT) B-form, i.e. what they called the Xform (11,12). In addition, other CD measurements yielded that it is possible to induce a clearly cooperative A-to-X transition in poly(dA-dT) (by millimolar concentrations ofCsCl): this proved that the X form is not merely a variant of the Aeonformation (13). In the same paper, the authors gave additional proofs of the discrepancy between the A form of poly(dA-dT) on one hand and the X forms of poly(dA-dT) (13) and poly(d2NH A-dT)(9) on the other, based on 31 P NMR arguments. Thus the close similarities ofnp NMR and CD features ofboth polynucleotides in their highsalt forms suggested that the salt- or alcohol-induced form ofpoly(d2NH2A-dT) is in fact the double helix in the X conformation. The present paper aims to clarify the problematic transition of poly(d2NH2A-dT), by using advantages provided by resonance Raman spectroscopy (RRS). Raman spectroscopy is known to be a valuable experimental method complementary to Xray, CD and NMR spectroscopies, extensively used in the two past decades for conformational studies of nucleic acid secondary structures (see references in (14)). At present, the main DNA families are fairly well characterized by their non-resonance Raman spectra and numerous so-called Raman conformational markers have been determined for A, B-, and Z-DNA( 14-19). In RRS the resonance enhancement of some vibrational lines is observed via excitation in the electronic transitions of the purine and pyrimidine bases : it offers additional possibilities, due to an increased sensitivity to the changes in the base stacking interactions resulting from any transitions. The application of RRS to DNA studies has recently been thoroughly reviewed (20).
Experimental Double stranded poly(d2NH 2A-dT), synthesized from d2NH 2ATP and dTTP using poly(dA-dT) as a primer template (9), was a generous gift from Dr. J. Sagi (Hungarian Academy ofSciences). Lyophilized polynucleotide was dissolved in 2.4 mM sodium phosphate I 0.3 mM EDTA buffer, pH 7.0, 0.1 M total concentration ofNa +cations, to obtain a stock solution of approximately 3.2 mM base pair concentration. The low-salt (0.1 M N a+) and medium-salt (1.6 M N a+) solutions of poly(d2NH0--dT) were prepared by mixing equal volumes of the stock solution with 0.1 M N aCl and 3.1 M NaCl buffers, respectively. The high-salt solution (4.0 M Na +)was obtained from the medium-salt one by slow addition of a proper amount of solid NaCl. Finally all three samples were in the same concentration in base pairs, i.e. 1.6 mM. The conformations ofpoly(d2NH2A-dT) in the low- and high-salt solutions correspond to initial and final states of the cooperative transition, respectively, with a midpoint occurring at 1.55 M NaCl (2). The conformation of each sample was tested with a Jobin-Yvon Mark V dichrograph before and after Raman measurements. The CD spectra were found identical to those of the results already published (2,9), and no change was detected after laser irradiation.
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ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy Peter Mojzes , Laurent Chinsky , Pierre-Yves Turpin & Pavol Miskovsky To cite this article: Peter Mojzes , Laurent Chinsky , Pierre-Yves Turpin & Pavol Miskovsky (1992) Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy, Journal of Biomolecular Structure and Dynamics, 10:1, 181-194, DOI: 10.1080/07391102.1992.10508637 To link to this article: http://dx.doi.org/10.1080/07391102.1992.10508637
Published online: 21 May 2012.
Submit your article to this journal
Article views: 3
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbsd20 Download by: [New York University]
Date: 15 November 2015, At: 06:06
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 10, Issue Number 1 (1992), "'Adenine Press (1992).
Downloaded by [New York University] at 06:06 15 November 2015
Salt-Induced Conformational Transition of Poly(d2NH2A-dT) Studied by Ultraviolet Resonance Raman Spectroscopy Peter Mojzes2 Laurent Chinsky1, Pierre-Yves Turpini,• and Pavol Miskovsky3 1
L.P.C.B. (CNRS UA 198) Institut Curie and Universite P. et M. Curie 75231 Paris Cedex 05, France 2
Institute of Physics Charles University CS-12116 Prague 2, Czechoslovakia 3
Department of Biophysics Safarik University CS-04054 Kosice, Czechoslovakia Abstract The conformational changes of poly( d2NH2A-dT) in aqueous solution, induced by increasing the NaCI concentration from O.IM to 4M, have been monitored by ultraviolet resonance Raman spectroscopy, in using the 222-, 257- and 281 nm excitation wavelengths. These changes have been interpreted in comparing the polymer spectra to those of the mononucleotide compounds on one hand, and to those of other alternating purine-pyrimidine polymers on the other hand, i.e. poly(dG-dC) and poly( dA-dT) which showed a B to Z transition in going from low- to high salt concentrations. The high salt poly( d2NH2A-dT) spectra do not show any Raman marker line of the Z conformation. The spectroscopic results indicate that most ofthe ribose puckering goes from C2' -endo/anti to C3' -endo/anti in increasing the salt concentration. In addition the base stacking interactions, to which the resonance Raman effect is very sensitive, are not drastically changed upon salt variations. Thus the high salt structure of poly( d2NH 2A-dT) remains a right-handed helix, likely under a dominant A conformation.
Introduction The salt-induced conformational transition of a self-complementary hexanucleotide consisting of deoxythymidine (d1) and 2-aminodeoxyadenosine (d2NH:zA), i.e. (dT-d2NH2A)3, has been at first monitored by CD spectroscopy ( 1). The authors proposed that it was similar to a B-Z transition (rigth to left-handed helix), on the • Author to whom correspondence should be addressed.
181
Downloaded by [New York University] at 06:06 15 November 2015
182
Mojzes et al.
basis of similarities with the B-Z transition of poly(dG-dC), i.e. essentially the sign inversion of the first CD extremum which is usually correlated to the inversion of helix handedness. The same cooperative transition, induced by a gradual increase ofNaCl concentration, has been observed in aqueous solutions of(d2NH2A-dT)n oligomers (2) and poly(d2NH2A-dT) alternating copolymers (3}, and monitored by CD, IR, UV-visible and 31 P NMR spectroscopies. The CD results could again be interpreted in terms ofB-Z transitions. Moreover, this was supported by the resemblances between the 2NH~ and guanine molecules, due to the 2-amino substitution in adenine : as in the case of in poly(dG-dC), this bulky amino group in the minor groove of poly( d2NH~-dT) , disrupting a regular spine of hydration, was supposed to be the crucial factor destabilizing the rigth-handed B-ONA. thus allowing a transition to the left-handed Z form (4). This was correlated to the sign inversion of poly(d2NH2A-dT) high-salt CD spectra (3), in opposition to what is observed in poly(dA-dT} CD spectra under the same salt conditions. However Howard et al. (2) showed thatthe 31 P NMR spectrum oflow-salt (d2NH2AdT}n consisted in two resonances of approximately equal intensity, separated by 32 Hz. The same doublet (separation of24 Hz) had been observed for low-salt poly(dAdT} (5) and assigned to an alternating conformation (alternating B-ONA) of the phosphodiester backbone of this polymer in solution. On increasing the salt concentration in poly(d2NH~-dT) solution, the separation of these two resonances increased up to 71 Hz, along with a downfield shift (2). This suggests further differentiation between two distinct phosphorus environments in the phosphodiester backbone, probably in an alternating array. The changes bear a cooperative character, suggesting a transition between two distinct alternating conformations. In addition, the interpretation based on CD measurements has been questioned by 20 NOE proton NMR results (6,7), which established that the conformation ofboth d2NH2A and dT residues in the high-salt form ofpoly(d2NH2A-dT) is C3'-endo/ anti, whereas the purine nucleosides in Z-ONA should be C3'-endo/syn. Consequently, Borah et al. concluded thatthe polynucleotide conformation would neither be the Z nor the B form, but instead an A form or closely related structure (6) having pyrimidine as well as purine residues in the C3'-endo/anti conformation. On the other hand, preliminary X-ray diffraction results on poly( d2NH~-dT) salt fibers (at 98% r.h.) have been recently published (8). In terms of overall distribution of intensities, the diffraction patterns suggest a structure which is close to that of ADNA. although the helical periodicity (12 base pairs per tum) and the crystal packing appreciably differ from those of A-DNA. and from those of other known deoxyribonucleodide structures as well. Moreover, the handedness ofthe poly(d2NH2AdT) helix in salt fibers was not established, thus the question whether the poly(d2NH~ dT) undergoes inversion of its handedness on salt-induced transition still remains open. In recent CD studies, Vorlickova et al. have shown that the conformation induced by various alcohols (9) or by sub millimolar concentrations ofMg 2+ cations (10) in low-salt poly(d2NH2A-dT) aqueous solutions is similar to that of the high-salt solution
Downloaded by [New York University] at 06:06 15 November 2015
Conformational Transition of Poly(d2NH~·dT)
183
of the same polymer. They also observed strong similarities between the high-salt form of poly(d2NH0--dT) and what they had observed in increasing drastically the CsF concentration in a solution of poly(dA-dT) B-form, i.e. what they called the Xform (11,12). In addition, other CD measurements yielded that it is possible to induce a clearly cooperative A-to-X transition in poly(dA-dT) (by millimolar concentrations ofCsCl): this proved that the X form is not merely a variant of the Aeonformation (13). In the same paper, the authors gave additional proofs of the discrepancy between the A form of poly(dA-dT) on one hand and the X forms of poly(dA-dT) (13) and poly(d2NH A-dT)(9) on the other, based on 31 P NMR arguments. Thus the close similarities ofnp NMR and CD features ofboth polynucleotides in their highsalt forms suggested that the salt- or alcohol-induced form ofpoly(d2NH2A-dT) is in fact the double helix in the X conformation. The present paper aims to clarify the problematic transition of poly(d2NH2A-dT), by using advantages provided by resonance Raman spectroscopy (RRS). Raman spectroscopy is known to be a valuable experimental method complementary to Xray, CD and NMR spectroscopies, extensively used in the two past decades for conformational studies of nucleic acid secondary structures (see references in (14)). At present, the main DNA families are fairly well characterized by their non-resonance Raman spectra and numerous so-called Raman conformational markers have been determined for A, B-, and Z-DNA( 14-19). In RRS the resonance enhancement of some vibrational lines is observed via excitation in the electronic transitions of the purine and pyrimidine bases : it offers additional possibilities, due to an increased sensitivity to the changes in the base stacking interactions resulting from any transitions. The application of RRS to DNA studies has recently been thoroughly reviewed (20).
Experimental Double stranded poly(d2NH 2A-dT), synthesized from d2NH 2ATP and dTTP using poly(dA-dT) as a primer template (9), was a generous gift from Dr. J. Sagi (Hungarian Academy ofSciences). Lyophilized polynucleotide was dissolved in 2.4 mM sodium phosphate I 0.3 mM EDTA buffer, pH 7.0, 0.1 M total concentration ofNa +cations, to obtain a stock solution of approximately 3.2 mM base pair concentration. The low-salt (0.1 M N a+) and medium-salt (1.6 M N a+) solutions of poly(d2NH0--dT) were prepared by mixing equal volumes of the stock solution with 0.1 M N aCl and 3.1 M NaCl buffers, respectively. The high-salt solution (4.0 M Na +)was obtained from the medium-salt one by slow addition of a proper amount of solid NaCl. Finally all three samples were in the same concentration in base pairs, i.e. 1.6 mM. The conformations ofpoly(d2NH2A-dT) in the low- and high-salt solutions correspond to initial and final states of the cooperative transition, respectively, with a midpoint occurring at 1.55 M NaCl (2). The conformation of each sample was tested with a Jobin-Yvon Mark V dichrograph before and after Raman measurements. The CD spectra were found identical to those of the results already published (2,9), and no change was detected after laser irradiation.
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