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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes a
T. J. Thomas & Thresia Thomas a
b
Departments of Medicine
b
Departments of Environmental and Community Medicine , University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School , New Brunswick , New Jersey , 08903 Published online: 21 May 2012.
To cite this article: T. J. Thomas & Thresia Thomas (1990) Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes, Journal of Biomolecular Structure and Dynamics, 7:6, 1221-1235, DOI: 10.1080/07391102.1990.10508561 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508561
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [New York University] at 00:22 19 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 7, Issue Number 6 (1990), "'Adenine Press (1990).
Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes
Downloaded by [New York University] at 00:22 19 May 2015
T.J. Thomas 1 and Thresia Thomas 2
Departments of 1Medicine and 2Environmental and Community Medicine University of Medicine and Dentistry of New JerseyRobert Wood Johnson Medical School New Brunswick, New Jersey 08903 Abstract We studied the effects of hexammine and tris( ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH 3) 63+ provoked a B-DNA-+ Z-DNA-+ '1'-DNA conformational transition in poly( dG-dC) · poly( dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the 'I'-DNA form. In addition, we compared the efficacy of Rh(NH3)63+ and Rh(en)r to provoke the Z-DNA conformation in poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC). The concentrations ofRh(NH 3 and Rh(en)/+ at the midpoint B-DNA-+ Z-DNA transition ofpoly(dG-dC) · poly(dG-dC) were 48 ± 2 and 238 ± 2 J.1M, respectively. The '1'-DNA form ofpoly(dG-dC) · poly(dG-dC) was stabilized at 500 11M Rh(NH 3) 6H. With poly(dG-m5dC) · poly(dG-m5dC), both counterions provoked the Z-DNA form at approximately 5 11M and stabilized the polynucleotide in this form up to 1000 11M concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly( dG-dC) · poly( dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.
)r
Introduction Inorganic complexes of the Group VIIIB transition metals play an important role in the stabilization, condensation and conformational transitions of native DNAs and synthetic polynucleotides (1-5). Behe and Felsenfeld (6) showed that micromolar concentrations of Co(NH 3) 63+ were capable of provoking the left-handed Z-ONA conformation ofpoly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dGm5 dC). This was an important discovery because until then it was believed that the
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presence of molar concentrations of Na + or dehydrating conditions were a prerequisite for the formation of Z-DNA conformation of poly(dG-dC) · poly(dG-dC) (7,8). Recent studies (5) also revealed that Ru(NH 3) 63+ is as effective as Co(NH3) 63 to induce the B-DNA to Z-DNA transition of poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) ·poly(dG-m 5dC). Even though circular dichroism (CD) spectroscopic technique could not detect aggregation or condensation in Co(NH 3) 63+ -induced ZDNA (6), Thomas and Bloomfield (9) showed from quasi-elastic laser light scattering and electron microscopic studies that Co(NH3) 63+ -induced Z-DNA conformational transition of poly(dG-m5dC) · poly(dG-m5dC) is accompanied by the collapse of the polynucleotide from its extended worm-like structure to a dense porulation oftoroids. This result is compatible with other reports showing that Co(NH3)6 + is capable of inducing the condensation of native viral DNAs (4,10). Eichhorn et al. (11) showed that Co(NH 3) 6 3+ is more than a Z-DNA inducer of poly(dG-dC) · poly(dG-dC). In a series of experiments, they provided evidence for the formation of a '1'-DNA structure of this polynucleotide at concentrations higher than that required to induce the Z-DNAform (11,12). The '1'-DNAconformation is believed to be a twisted, tightly packaged assembly of DNA (13,14). The handedness of this structure had been under controversy for some time (12). Using a monoclonal antiZ-DNA antibody, we (15) recently provided direct evidence that the Co(NH 3) 63+induced 'I'- DNA form of the polynucleotides exists in the left-handed Z-DNA conformation. In order to further understand the structural dynamics of polynucleotides in the presence of metal complexes, we undertook the present study to examine the effects ofRh(NH3) 63+ and Rh( en)/+ on the conformation of poly(dGdC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC). We also addressed the question of ligand specificity in the efficacy of metal complexes to provoke the BDNA to Z-DNA transition in polynucleotides. Using ultraviolet (UV) and CD spectroscopy and an enzyme immunoassay to detect the presence ofZ-DNA conformation, we demonstrate that Rh(NH 3) 63+ and Rh( en)33+ are capable of provoking a series of sequential conformational transitions in these polynucleotides. Materials and Methods Polynucleotides
Poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC) were purchased from Pharmacia, Inc. (Piscataway, NJ). The polynucleotides were dissolved in a buffer containing 50 mM NaCI, I mM Na cacodylate, 0.15 mM EDTA, pH 7.4 and dialyzed extensively from the same buffer using a Spectrapor membrane tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) with a molecular weight cut off of6,000-8,000. Stock solutions of the polynucleotides were prepared at 0.75 mglml and stored at4 oc. The stock solutions were diluted to 10 JJg/ml for use in the enzyme immunoassays. The optical density of the polynucleotides was measured with a Beckman DU-8B spectrophotometer. Molar concentrations were calculated using the molar extinction coefficients of 8,400 and 6,800 for poly( dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC), respectively (16). For some control experiments, we used brominated poly(dG-dC) · poly(dG-dC) that was prepared by the method described by Lafer et a/. ( 17).
Effect of Rhodium on Polynucleotide Conformations
1223
Rhodium Complexes
Hexamminerhodium(III) chloride and tris (ethylenediamine)rhodium(III) chloride were purchased from Johnson Mathey, Inc. (Seabrook, NH). The complexes were purified by recrystallization from ethanol. Stock solutions of these compounds were prepared in double distilled, deionized water at a high concentration to keep the volume of the counterion solutions added to the polynucleotide at less than 3% of the total volume of solution. The concentrations of the complexes were calculated from the followin:ft values of their extinction(~>) coefficients: Rh(NH 3)/+ (307 nm): 134, and Rh(en)3 + (301 nm): 249.
Downloaded by [New York University] at 00:22 19 May 2015
Monoclonal Anti-Z-DNA Antibody Z22
The monoclonal antibody Z22 used in these studies was a kind gift of B. David Stollar, M.D., Professor and Chairman, Department ofBiochemistry, Tufts University, Boston, MA. This antibody was prepared by the hybridoma technique (18,19). In summary, C57Bl/6 mouse was immunized with brominated poly(dG-dC) · poly(dGdC) and the spleen cells were fused with SP2/0-Agl4, a nonsecreting myeloma. We obtained the antibody from hybridoma culture supernatants at a concentration of350 11g/ml in phosphate buffered saline (PBS: O.ol M phosphate, 0.15 M NaCl, pH 7.4). Aliquots of the stock solution were diluted in PBS containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate) and 0.02% NaN 3 for the enzyme immunoassays. Immunochemicals
Protamine sulfate, alkaline phosphatase-conjugated, affinity purified polyvalent goat anti-mouse immunoglobulins and phosphatase substrate (p-nitrophenyl phosphate) were purchased from Sigma Chemical Company. We used Costar polystyrene 96-well microtiter plates (cat. #3590) (Costar, Cambridge, MA) as the solid phase for immobilizing the conformations of the polynucleotides in the presence of metal complexes. Spectroscopic Studies
UV spectra of the polynucleotides and their combinations with metal complexes were determined with a Beckman DU-8B spectrophotometer. In experiments with metal complexes and polynucleotides, we used a blank solution containing identical concentration of metal complexes to subtract the contribution of the spectra of metal complexes from that of metal complex-polynucleotide combinations. The BDNA to Z-DNA transition of the polynucleotides was monitored by recording the absorbance ratio (~w/A295 ) at different concentrations of the counterions. In the BDNA conformation, A26ofA295 ratios of poly(dG-dC) · poly(dG-dC) and poly( dGm5dC) · poly(dG-m5dC) are 7.6 ± 0.4 and 4.2 ± 0.2, respectively (9). In the Z-DNA conformation, the corresponding ratios are 4.0 ± 0.2 and 2.6 ± 0.1. The CD spectra of poly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dG-m5dC) were recorded with a Jasco J41 spectropolarimeter. The molar ellipticity was calculated
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from the equation [9] = 9/cl, where [9] is the molar ellipticity, 9 is the relative intensity, c is the molar concentration of polynucleotides, and 1is the path length of the cell in centimeters. In CD spectroscopy, the inversion of the spectrum was taken as an indication ofB-DNA to Z-DNA transition. The intensity of the spectral peak at 292 nm was used as a marker of the conformational transitions of the polynucleotides. On B-DNA to zDNA transition, this peak changed from a positive value to a strong negative value. In all spectroscopic measurements; the polynucleotides were mixed with the necessary concentrations of metal complexes and incubated for 1 h to attain equilibrium. The UV and CD spectra were recorded in the wavelength range of220 to 350 nm. The concentration of metal complexes at the midpoint ofB-DNA to Z-DNA transition was determined byplottingA26d'A 295 and (9lz92 nm againstthe concentration of the counterion in UV and CD spectroscopy, respectively.
Enzyme Immunoassay Protocol We coated microtiter plates with 0.3 ml per well 0.0001% solution of protamine sulfate to facilitate the adsorption of the polynucleotides to the polystyrene surface (20). The microtiter plates were incubated for 90 min at room temperature and washed 3 times with double distilled, deionized water using a Perkin Elmer-Cetus PRO/PETIE instrument with a PRO/WASH head or a Beckman 1000 Automated Laboratory Workstation. The plates were then treated with 0.2 ml per well of poly(dG-dC) · poly(dG-dC) or poly(dGm5 dC) · poly(dG-m5dC) that was previously incubated with the necessary concentrations of metal complexes. Polynucleotide solutions used in this study had an absotbance of0.15 O.D. at 260 nm. In order to achieve a monolayer of the polynucleotide on the surface of the microtiter wells, and to attain equilibrium of the reaction between the polynucleotides and the metal complexes, we incubated the plates for 16 hat 4 oc. The plates were washed 3 times with PBS-Tween 20-NaN3. After the washings, the plates were treated with 0.2 ml per well of monoclonal antibody Z22 at a concentration of0.35 !Jg/ml in PBS-Tween 20-NaN3 buffer and incubated for 1 h at 37°C. The plates were washed 3 times with PBS-Tween 20-NaN3 and further treated with 0.2 ml per well of alkaline phosphatase-conjugated goat anti-mouse polyvalent immunoglobulins (Sigma) diluted 1:350in PBS-Tween20-NaN3. Theplateswereincubatedfor 1 h at37°C, washed with PBS-Tween 20-NaN3 and treated with 0.2 ml of a solution of phosphatase substrate (p-nitrophenyl phosphate) at a concentration of 1 mg per ml in 0.05 M sodium bicarbonate, 0.001 M MgC1 2 and 0.02% NaN 3, pH 9.0. The plates were incubated at room temperature in the dark for 30 min to complete the enzymatic reaction. The enzymesubstrate reaction was stopped by the addition of0.05 ml of 1 N NaOH solution. The optical density of the solutions in the microtiter wells was measured at 405 nm with a Biotek (Winooski, Vf) EL 309 Microplate Autoreader or a Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA). These instruments have a useful optical density range of 0 to 2.99 O.D. units.
Results Ultraviolet Spectroscopy In order to understand the ability ofRh(NH3) 63+ and Rh(en)/+ to induce the Z-DNA
Effect of Rhodium on Polynucleotide Conformations
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a.o-------------·
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0
-
1a'Q)
0
c
cu
-e0 (/J
~
Concentration of Rh complexes, JlM Figure 1: The absorbance ratio (A2ro/Af95 ) of poly(dG-dC) · poly(dG-dC) plotted against the concentrations ofRh(NH 3) 6H (.£1) and Rh(en)3 +(e).
conformation in two polynucleotides that are known to assume the Z-DNA form under a variety of conditions, we recorded the UV spectra of poly(dG-dC) · poly(dGdC) and poly(dG-m5dC) · poly(dG-m5dC) in the presence of different concentrations of these complexes. Since the ratio of absorbance (A) at 260 to 295 nm is a marker of the B-DNA and Z-DNA conformations of these polynucleotides, we determined these ratios and plotted them against the corresponding concentration of metal complexes. Figure 1 shows the A2r,olA295 values of poly( dG-dC) · poly(dGdC) plotted against the concentration of metal complexes. At low concentrations of Rh(NH 3) 6 3+, A2r,o/A295 ofpoly(dG-dC) · poly(dG- dC) remained at 7.6 ± 0.4. This value is characteristic of the B-DNA conformation. In the presence of 20 fJM and above of Rh(NH 3) 63+, there was a continuous decrease in the value of A26ofA295 ratio. In contrast toN a+ -induced B-DNA to z- DNA transition, where the A26ofA295 ratio of the polynucleotide leveled off at 3M NaCl (7,9), there was no plateau region in A2&JA295 ratio of poly(dG-dC) · poly(dG-dC) at high concentrations ofRh(NH 3) 63+. Instead, a continuous decrease of this ratio was observed at all concentrations of metal complexes that we studied. Therefore, we could not determine a midpoint
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4.4r----------------...,
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en
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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes a
T. J. Thomas & Thresia Thomas a
b
Departments of Medicine
b
Departments of Environmental and Community Medicine , University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School , New Brunswick , New Jersey , 08903 Published online: 21 May 2012.
To cite this article: T. J. Thomas & Thresia Thomas (1990) Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes, Journal of Biomolecular Structure and Dynamics, 7:6, 1221-1235, DOI: 10.1080/07391102.1990.10508561 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508561
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [New York University] at 00:22 19 May 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, /SSN 0739-1102 Volume 7, Issue Number 6 (1990), "'Adenine Press (1990).
Conformational Transitions of Polynucleotides in the Presence of Rhodium Complexes
Downloaded by [New York University] at 00:22 19 May 2015
T.J. Thomas 1 and Thresia Thomas 2
Departments of 1Medicine and 2Environmental and Community Medicine University of Medicine and Dentistry of New JerseyRobert Wood Johnson Medical School New Brunswick, New Jersey 08903 Abstract We studied the effects of hexammine and tris( ethylene diamine) complexes of rhodium on the conformation of poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC) using spectroscopic techniques and an enzyme immunoassay. Circular dichroism spectroscopic measurements showed that Rh(NH 3) 63+ provoked a B-DNA-+ Z-DNA-+ '1'-DNA conformational transition in poly( dG-dC) · poly( dG-dC). Using the enzyme immunoassay technique with a monoclonal anti-Z-DNA antibody, we found that the left-handedness of the polynucleotide was maintained in the 'I'-DNA form. In addition, we compared the efficacy of Rh(NH3)63+ and Rh(en)r to provoke the Z-DNA conformation in poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC). The concentrations ofRh(NH 3 and Rh(en)/+ at the midpoint B-DNA-+ Z-DNA transition ofpoly(dG-dC) · poly(dG-dC) were 48 ± 2 and 238 ± 2 J.1M, respectively. The '1'-DNA form ofpoly(dG-dC) · poly(dG-dC) was stabilized at 500 11M Rh(NH 3) 6H. With poly(dG-m5dC) · poly(dG-m5dC), both counterions provoked the Z-DNA form at approximately 5 11M and stabilized the polynucleotide in this form up to 1000 11M concentration. These results show that trivalent complexes of Rh have a profound influence on the conformation of poly( dG-dC) · poly( dG-dC) and its methylated derivative. Furthermore, the Rh complexes are capable of maintaining the Z-DNA form at concentration ranges far higher than that of other trivalent complexes. Our results also demonstrate that the efficacy of trivalent inorganic complexes to induce the B-DNA to Z-DNA transition of poly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dG-m5dC) is dependent on the nature of the ligand as well as the polynucleotide modification. Differences in charge density and hydration levels of counterions or base sequence- and counterion-dependent specific interactions between DNA and metal complexes might be possible mechanisms for the observed effects.
)r
Introduction Inorganic complexes of the Group VIIIB transition metals play an important role in the stabilization, condensation and conformational transitions of native DNAs and synthetic polynucleotides (1-5). Behe and Felsenfeld (6) showed that micromolar concentrations of Co(NH 3) 63+ were capable of provoking the left-handed Z-ONA conformation ofpoly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dGm5 dC). This was an important discovery because until then it was believed that the
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presence of molar concentrations of Na + or dehydrating conditions were a prerequisite for the formation of Z-DNA conformation of poly(dG-dC) · poly(dG-dC) (7,8). Recent studies (5) also revealed that Ru(NH 3) 63+ is as effective as Co(NH3) 63 to induce the B-DNA to Z-DNA transition of poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) ·poly(dG-m 5dC). Even though circular dichroism (CD) spectroscopic technique could not detect aggregation or condensation in Co(NH 3) 63+ -induced ZDNA (6), Thomas and Bloomfield (9) showed from quasi-elastic laser light scattering and electron microscopic studies that Co(NH3) 63+ -induced Z-DNA conformational transition of poly(dG-m5dC) · poly(dG-m5dC) is accompanied by the collapse of the polynucleotide from its extended worm-like structure to a dense porulation oftoroids. This result is compatible with other reports showing that Co(NH3)6 + is capable of inducing the condensation of native viral DNAs (4,10). Eichhorn et al. (11) showed that Co(NH 3) 6 3+ is more than a Z-DNA inducer of poly(dG-dC) · poly(dG-dC). In a series of experiments, they provided evidence for the formation of a '1'-DNA structure of this polynucleotide at concentrations higher than that required to induce the Z-DNAform (11,12). The '1'-DNAconformation is believed to be a twisted, tightly packaged assembly of DNA (13,14). The handedness of this structure had been under controversy for some time (12). Using a monoclonal antiZ-DNA antibody, we (15) recently provided direct evidence that the Co(NH 3) 63+induced 'I'- DNA form of the polynucleotides exists in the left-handed Z-DNA conformation. In order to further understand the structural dynamics of polynucleotides in the presence of metal complexes, we undertook the present study to examine the effects ofRh(NH3) 63+ and Rh( en)/+ on the conformation of poly(dGdC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC). We also addressed the question of ligand specificity in the efficacy of metal complexes to provoke the BDNA to Z-DNA transition in polynucleotides. Using ultraviolet (UV) and CD spectroscopy and an enzyme immunoassay to detect the presence ofZ-DNA conformation, we demonstrate that Rh(NH 3) 63+ and Rh( en)33+ are capable of provoking a series of sequential conformational transitions in these polynucleotides. Materials and Methods Polynucleotides
Poly(dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC) were purchased from Pharmacia, Inc. (Piscataway, NJ). The polynucleotides were dissolved in a buffer containing 50 mM NaCI, I mM Na cacodylate, 0.15 mM EDTA, pH 7.4 and dialyzed extensively from the same buffer using a Spectrapor membrane tubing (Spectrum Medical Industries, Inc., Los Angeles, CA) with a molecular weight cut off of6,000-8,000. Stock solutions of the polynucleotides were prepared at 0.75 mglml and stored at4 oc. The stock solutions were diluted to 10 JJg/ml for use in the enzyme immunoassays. The optical density of the polynucleotides was measured with a Beckman DU-8B spectrophotometer. Molar concentrations were calculated using the molar extinction coefficients of 8,400 and 6,800 for poly( dG-dC) · poly(dG-dC) and poly(dG-m 5dC) · poly(dG-m 5dC), respectively (16). For some control experiments, we used brominated poly(dG-dC) · poly(dG-dC) that was prepared by the method described by Lafer et a/. ( 17).
Effect of Rhodium on Polynucleotide Conformations
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Rhodium Complexes
Hexamminerhodium(III) chloride and tris (ethylenediamine)rhodium(III) chloride were purchased from Johnson Mathey, Inc. (Seabrook, NH). The complexes were purified by recrystallization from ethanol. Stock solutions of these compounds were prepared in double distilled, deionized water at a high concentration to keep the volume of the counterion solutions added to the polynucleotide at less than 3% of the total volume of solution. The concentrations of the complexes were calculated from the followin:ft values of their extinction(~>) coefficients: Rh(NH 3)/+ (307 nm): 134, and Rh(en)3 + (301 nm): 249.
Downloaded by [New York University] at 00:22 19 May 2015
Monoclonal Anti-Z-DNA Antibody Z22
The monoclonal antibody Z22 used in these studies was a kind gift of B. David Stollar, M.D., Professor and Chairman, Department ofBiochemistry, Tufts University, Boston, MA. This antibody was prepared by the hybridoma technique (18,19). In summary, C57Bl/6 mouse was immunized with brominated poly(dG-dC) · poly(dGdC) and the spleen cells were fused with SP2/0-Agl4, a nonsecreting myeloma. We obtained the antibody from hybridoma culture supernatants at a concentration of350 11g/ml in phosphate buffered saline (PBS: O.ol M phosphate, 0.15 M NaCl, pH 7.4). Aliquots of the stock solution were diluted in PBS containing 0.05% Tween 20 (polyoxyethylene sorbitan monolaurate) and 0.02% NaN 3 for the enzyme immunoassays. Immunochemicals
Protamine sulfate, alkaline phosphatase-conjugated, affinity purified polyvalent goat anti-mouse immunoglobulins and phosphatase substrate (p-nitrophenyl phosphate) were purchased from Sigma Chemical Company. We used Costar polystyrene 96-well microtiter plates (cat. #3590) (Costar, Cambridge, MA) as the solid phase for immobilizing the conformations of the polynucleotides in the presence of metal complexes. Spectroscopic Studies
UV spectra of the polynucleotides and their combinations with metal complexes were determined with a Beckman DU-8B spectrophotometer. In experiments with metal complexes and polynucleotides, we used a blank solution containing identical concentration of metal complexes to subtract the contribution of the spectra of metal complexes from that of metal complex-polynucleotide combinations. The BDNA to Z-DNA transition of the polynucleotides was monitored by recording the absorbance ratio (~w/A295 ) at different concentrations of the counterions. In the BDNA conformation, A26ofA295 ratios of poly(dG-dC) · poly(dG-dC) and poly( dGm5dC) · poly(dG-m5dC) are 7.6 ± 0.4 and 4.2 ± 0.2, respectively (9). In the Z-DNA conformation, the corresponding ratios are 4.0 ± 0.2 and 2.6 ± 0.1. The CD spectra of poly(dG-dC) · poly(dG-dC) and poly(dG-m5dC) · poly(dG-m5dC) were recorded with a Jasco J41 spectropolarimeter. The molar ellipticity was calculated
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from the equation [9] = 9/cl, where [9] is the molar ellipticity, 9 is the relative intensity, c is the molar concentration of polynucleotides, and 1is the path length of the cell in centimeters. In CD spectroscopy, the inversion of the spectrum was taken as an indication ofB-DNA to Z-DNA transition. The intensity of the spectral peak at 292 nm was used as a marker of the conformational transitions of the polynucleotides. On B-DNA to zDNA transition, this peak changed from a positive value to a strong negative value. In all spectroscopic measurements; the polynucleotides were mixed with the necessary concentrations of metal complexes and incubated for 1 h to attain equilibrium. The UV and CD spectra were recorded in the wavelength range of220 to 350 nm. The concentration of metal complexes at the midpoint ofB-DNA to Z-DNA transition was determined byplottingA26d'A 295 and (9lz92 nm againstthe concentration of the counterion in UV and CD spectroscopy, respectively.
Enzyme Immunoassay Protocol We coated microtiter plates with 0.3 ml per well 0.0001% solution of protamine sulfate to facilitate the adsorption of the polynucleotides to the polystyrene surface (20). The microtiter plates were incubated for 90 min at room temperature and washed 3 times with double distilled, deionized water using a Perkin Elmer-Cetus PRO/PETIE instrument with a PRO/WASH head or a Beckman 1000 Automated Laboratory Workstation. The plates were then treated with 0.2 ml per well of poly(dG-dC) · poly(dG-dC) or poly(dGm5 dC) · poly(dG-m5dC) that was previously incubated with the necessary concentrations of metal complexes. Polynucleotide solutions used in this study had an absotbance of0.15 O.D. at 260 nm. In order to achieve a monolayer of the polynucleotide on the surface of the microtiter wells, and to attain equilibrium of the reaction between the polynucleotides and the metal complexes, we incubated the plates for 16 hat 4 oc. The plates were washed 3 times with PBS-Tween 20-NaN3. After the washings, the plates were treated with 0.2 ml per well of monoclonal antibody Z22 at a concentration of0.35 !Jg/ml in PBS-Tween 20-NaN3 buffer and incubated for 1 h at 37°C. The plates were washed 3 times with PBS-Tween 20-NaN3 and further treated with 0.2 ml per well of alkaline phosphatase-conjugated goat anti-mouse polyvalent immunoglobulins (Sigma) diluted 1:350in PBS-Tween20-NaN3. Theplateswereincubatedfor 1 h at37°C, washed with PBS-Tween 20-NaN3 and treated with 0.2 ml of a solution of phosphatase substrate (p-nitrophenyl phosphate) at a concentration of 1 mg per ml in 0.05 M sodium bicarbonate, 0.001 M MgC1 2 and 0.02% NaN 3, pH 9.0. The plates were incubated at room temperature in the dark for 30 min to complete the enzymatic reaction. The enzymesubstrate reaction was stopped by the addition of0.05 ml of 1 N NaOH solution. The optical density of the solutions in the microtiter wells was measured at 405 nm with a Biotek (Winooski, Vf) EL 309 Microplate Autoreader or a Vmax kinetic microplate reader (Molecular Devices, Menlo Park, CA). These instruments have a useful optical density range of 0 to 2.99 O.D. units.
Results Ultraviolet Spectroscopy In order to understand the ability ofRh(NH3) 63+ and Rh(en)/+ to induce the Z-DNA
Effect of Rhodium on Polynucleotide Conformations
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Concentration of Rh complexes, JlM Figure 1: The absorbance ratio (A2ro/Af95 ) of poly(dG-dC) · poly(dG-dC) plotted against the concentrations ofRh(NH 3) 6H (.£1) and Rh(en)3 +(e).
conformation in two polynucleotides that are known to assume the Z-DNA form under a variety of conditions, we recorded the UV spectra of poly(dG-dC) · poly(dGdC) and poly(dG-m5dC) · poly(dG-m5dC) in the presence of different concentrations of these complexes. Since the ratio of absorbance (A) at 260 to 295 nm is a marker of the B-DNA and Z-DNA conformations of these polynucleotides, we determined these ratios and plotted them against the corresponding concentration of metal complexes. Figure 1 shows the A2r,olA295 values of poly( dG-dC) · poly(dGdC) plotted against the concentration of metal complexes. At low concentrations of Rh(NH 3) 6 3+, A2r,o/A295 ofpoly(dG-dC) · poly(dG- dC) remained at 7.6 ± 0.4. This value is characteristic of the B-DNA conformation. In the presence of 20 fJM and above of Rh(NH 3) 63+, there was a continuous decrease in the value of A26ofA295 ratio. In contrast toN a+ -induced B-DNA to z- DNA transition, where the A26ofA295 ratio of the polynucleotide leveled off at 3M NaCl (7,9), there was no plateau region in A2&JA295 ratio of poly(dG-dC) · poly(dG-dC) at high concentrations ofRh(NH 3) 63+. Instead, a continuous decrease of this ratio was observed at all concentrations of metal complexes that we studied. Therefore, we could not determine a midpoint
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