This article was downloaded by: [New York University] On: 06 December 2014, At: 14:25 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs a
a
Karsten Rippe , Niels B. Ramsing , Reinhard a
Klement & Thomas M. Jovin
a
a
Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology , P.O. Box 2841, D-3400 , Göttingen , F.R.G. Published online: 21 May 2012.
To cite this article: Karsten Rippe , Niels B. Ramsing , Reinhard Klement & Thomas M. Jovin (1990) A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs, Journal of Biomolecular Structure and Dynamics, 7:6, 1199-1209, DOI: 10.1080/07391102.1990.10508559 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508559
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 14:25 06 December 2014
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).
A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs
Downloaded by [New York University] at 14:25 06 December 2014
Karsten Rippe, Niels B. Ramsing t, Reinhard Klement and Thomas M. Jovin * Max Planck Institute for Biophysical Chemistry Department of Molecular Biology P.O. Box 2841 D-3400 Gottingen, F.R.G. Abstract DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix ofB-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D 1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-l6°C lower and the van't Hoff enthalpy difference AllvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgC12) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-(d(T5 G~) · d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.
Introduction Pairs of deoxyoligonucleotides consisting of dA and dT sequences designed to be complementary only if the strands are oriented with the same polarity hybridize spontaneously into stable double-helical structures (1-6). Distinctive structural features ofparallel stranded DNA(ps-DNA) derived from model calculations are reverse Watson-Crick base pairing with the glycosidic bonds in a trans orientation and the lack of distinct rnajar and minor grooves. Parallel stranded duplexes exhibit spectroscopic, thermodynamic, and biochemical properties different from those of conventional antiparallel B-ONA, and are remarkably stable (1-6). For example, the enthalpy change associated with the helix-coil transition ofps-DNAis only 1020% less than that of B-ONA (5,6), in qualitative agreement with the force field calculations ofPattabiraman who originally proposed the structure (7). All parallel t Current address:
Department of Ecology and Genetics, University of Arhus, Ny Munkegade, DK-8000 Arhus C, Denmark
*Author to whom correspondence should be addressed.
1199
Rippe eta/.
1200
stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. The potential biological significance of this alternative helical structure, however, depends upon its feasibility in the case of natural sequences containing dG and dC as well. Therefore we have substituted four dA · dT base pairs of a parallel stranded linear duplex with dG · dC base pairs and compared its properties to that of the ps reference duplex consisting exclusively of dA · dT base pairs. Materials and Methods
Downloaded by [New York University] at 14:25 06 December 2014
Oligonucleotide Synthesis and Characterization
e
The oligonucleotides were synthesized, purified, labelled with 2P], and hybridized as described previously (4). The purity of the isolated end-labelled oligonucleotides was established by electrophoresis under denaturing conditions (4). No contaminating faster or slower migrating species could be detected (data not shown). Gel electrophoresis under native conditions was carried out in 14% polyacrylamide (5% crosslinking), 90 mM Tris-borate, pH 8.0, 2 mM MgC12 and at 20°C. The nuclease susceptibilities of 32P-end labelled DNAs were assessed according to Ref. 8 with Aspergillus oryzae Sl nuclease (Pharmacia) and Staphylococcus aureus (Micrococcal) nuclease S7 (Sigma). Spectroscopy
UV absorption measurements were made with a Uvikon 820 spectrophotometer equipped with computer-controlled thermostated cuvette holders and data acquisition (5) in 0.4 M NaCl and 10 mM N a-cacodylate, pH 7.1, unless otherwise indicated. The molar extinction coefficient of the oligonucleotides D 1, D2, D3 and D4 was taken to be 8.6 mM (base) -I cm- 1 at 264 nm in the denatured state (70 C standard buffer+ 0.1 M NaCl; Figure 2B) (2,4). The corresponding extinction coefficients forDS and for D6 in the denatured state were estimated bycombiningthe dA · dTvaluecited above for D 1-4 with the extinction coefficient for poly [d(G-C)] in the native (9) and denatured (Ramsing &Jovin, in preparation) state, and the corresponding value for denatured poly[dC] (10). We calculated ane264 of8.6and8.4mmol base- 1 em-I forDS and D6, respectively, at 70°C. Circular dichroism (CD) spectra were acquired with a Jobin Yvon Model V Dichrograph in 2 mM MgC12, 10 mM Na-cacodylate, pH 7.1. Model Force Field Calculations
Energy minimization was with the AMBER-PMF program ( 11) using 0.1 M N aCl as the supporting electrolyte in conjunction with the INSIGHT (Biosym Tech.) molecular modelling package and a PS390 Evans and Sutherland graphics workstation. Results and Discussion Design of Oligonucleotides
The set of25-nt oligonucleotides D 1, D2, D3 and D4 shown below were designed by
1201
Parallel Stranded Linear DNA
Downloaded by [New York University] at 14:25 06 December 2014
a computer aided search for complementary sequences capable of forming parallel stranded (ps) helices with minimal interference from competitive antiparallel (aps) secondary structures (4). The two ps-DNA duplexes, ps-D 1 · D2, ps-D3 · D4, and the aps-Dl · D3 and aps-D2 · D4 references, were extensively characterized with respect to spectroscopic and thermodynamic properties (4-6) and were also tested as substrates for several DNA processing enzymes and chemical reagents (8). A comparison of the two combinations ps-D 1 · D2 and ps-D3 · D4 with the corresponding aps molecules revealed the same characteristic differences found previously with other ps-DNA constructs (1-6). In order to determine the influence of dG and dC residues on the properties of the parallel stranded helix, we synthesized two other 25-nt oligonucleotides designated as D5 and D6, in which four dA(D5) or four dT (D6) bases were replaced by dG and dC, respectively. Synthesis and purification were according to Ref. 4. The following combinations of the Dl-D6 oligonucleotides were stable (see below): PanlleliiDDcled duplexes
Anlipanllelltranded duplexes
01: 5 1 -AAAAAAAAAATAATTTTAAATATTT-3 1 5 I -TTTTTTTTTTATTAAAATTTATAAA-3 I :02 1
03: 5 -AAATATTTAAAATTATTTTTTTTTT-3
.............•••.........
1
5 I -TTTATAAATTTTAATAAAAAAAAAA-3 I :04 OS: 5 1 -AAAAAQAAAQTAQTTTTAAQTATTT-3 1 •••••••••a••••••••••••••• 5 I -TTTTTCTTTCATCAAAATTCATAAA-3 I :06
01: 5 1 -AAAAAAAAAATAATTTTAAATATTT-3 1 3 I -TTTTTTTTTTATTAAAATTTATAAA-5 I :03
........................ .
D2: 5 I -TTTTTTTTTTATTAAAATTTATAAA-3 I 3 I -AAAAAAAAAATAATTTTAAATATTT-5 I :04 ~: 5 1 -AAAAAQAAAGTAQTTTTAAQTATTT-3 1
••••.•...•..•......•.....
3 I -TTTTnTTT'fAT'fAAAATT'fATAAA-5 1 :03
Electrophoretic Properties The generation and stability of the proposed duplex structures were assessed by gel electrophoresis under native conditions (2 mM MgC1 2, 20°C; Figure lA). The only duplexes formed in addition to those described previously [ps-Dl · D2, ps-D3 · D4, aps-Dl · D3, aps-D2 · D4 (4,5)] corresponded to the combination D5+ D6 (designated ps-D5 · D6) that has a sequence with four dG · dC base pairs and complementarity in a parallel orientation, and D5+ D3 (antiparallel with four dG · dT mismatches). The other potential ps combinations involved either opposing dG and dT (D5+ D2) or dA and dC (Dl + D6) migrated in the position of single stranded species (with Dl + D6 showing a slightly lower mobility).
Spectroscopic Properties The ultraviolet absorption and circular dichroism spectra of the ps-D5 · D6 duplex are indicative of a base-paired structure resembling that of ps duplexes consisting onlyofdA · dTbasepairs. Thus, both ps-D5 · D6andps-Dl · D2 show a characteristic blue shift in the native UV spectra relative to aps-D 1 · D3 (Figure 2A), a feature particularly evident in the difference spectra (Figure 2B) and attributable to the dA · dT part of the sequence. Upon denaturation, the spectra (Figure 2B) demonstrate only the differences expected from the base composition. In Figure 2C the specific
Rippe eta/.
1202
Downloaded by [New York University] at 14:25 06 December 2014
dG · dC contribution in ps-05 · 06 was calculated by subtracting the fractional dA · dT absorbance estimated from the spectra of ps-01 · 02. In the denatured state, the specific dG · dC absorbance is similar to that of poly[d(G-C)] and to the estimated dG · dC contribution of poly d[(A-C) · d(G-T)]. In the native state, however, the calculated dG · dC spectrum ofps-05 · 06 exhibits a higher absorbance, a general broadening, and a red shift of the peak compared to the reference ONAs. [As noted in the legend to Figure 2C, perturbations of the adjoining dA · dT base pairs may also contribute.] TheCOspectrumofps-05 · 06comparedtothatoftheaps-Ol· 03referenceinthe native state (Figure 20) shows the same features observed with ps-01 · 02 above 265 nm (seen as a difference spectrum in Figure 2F) whereas at 250 nm the dichroism is reduced. The spectra in the denatured state are nearly similar for all three combinations (Figure 2E) showing only slight deviations attributable to the different base composition. Helix-Coil Transition
Salt-dependent thermal transitions of various duplexes were monitored by the increase of UV absorption upon melting and represented as three-dimensional plots (Figure 3A-E). The absorbances at each temperature T and wavelength were normalized to the values at the initial (low) temperature T0 in order to reveal the characteristic differences between the ps and aps conformation (2,4,5,6). The combination ps-05 · 06 in MgC12 or NaCl displays the same distinctive hyperchromicity pattern found with ps-01 · 02, ps-03 · 04 and otherps ONAs (2,4,5,6). The melting temperature Tm of ps-05 · 06 is 10-16°C lower than that of the corresponding duplex ps-01 · 02 composed only of dA · dT base pairs and has the same salt dependence as the reference ONAs (Figure 3F, Table I). The van't Hoff enthalpy difference tll/vH for the helix-coil transition of ps-05 · 06 is reduced by 20% (in NaCl) and 10% (in MgC12) compared to that ofps-01 · 02, although it is greater than the value determined for a 21-nt ps duplex consisting exclusively of dA · dT base pairs (2,5,6). We calculated the mean dG · dC contribution per mole of nearest neighbors (N.N.) ofps-05 · 06, ~hdG·dO by assuming that the substitution of the 4 dG · dC base pairs outof25leads to a change in 8 N.N. interactions. The resulting expression (~hdG. d~ = [tll/ps-os. 06 - 16/24 · tll/ps-Ot. 02 )18) yields 8 kJ ·mol-t in NaCl and 14 kJ · mol- in MgC12, respectively, and can be compared to the mean values for the
Legend for Color Folio Figure 4: Model of dG · dC base pair in ps(d(TsGA5) · d(A5CT5)] optimized by force field calculations. (A) Side-view of the central three base pairs; 5' ends are at the bottom. (B) eros-sectional view of the central dG · dC (ball and stick) and underlying dA · dT (space filling) base pair. Color coding: sugars, white; phosphates (including 03' and 05'), red; adenine, cyan; thymine, yellow; thymine methyl groups, turquoise; guanine, blue; cytosine, green. The figure was generated by the SCHAKAL v86b program of Dr. E. Keller, Univ. of Freiburg, FRG. The model was generated by extracting the 3' terminalll residues from an optimized 21-nt ps duplex (2) and refining the resultant 11-nt ps-(d(TsAt,) · d(A5T 6)] duplex as well as the sequence in which the central dA · dT base pair was replaced by dG · dC.
Downloaded by [New York University] at 14:25 06 December 2014
Downloaded by [New York University] at 14:25 06 December 2014
I ss
1,2 1,3 1,4 1,5 1,6 2 ps aps - ss
2,3 2,4 2,5 2,6 3 - aps - ss
3,4 3,5 3,6 4 ps - ss
4,5 4,6 - ss
5,6 6 ps ss
- - - - - - - - - - - - - native------------denaturing
- - - - - denaluring - - - - -
c
- - - - - S I nuclease---- - - - Micrococcus nMclease - - - - - 5,6-3,4-- - - - 5,6-- - - - 3,4-- - ps (G·C)- - - - ps - - - - - ps (G·C)- - - - ps - - 0 I 0 20 40 80 0 I 0 20 40 80 0 I 0 20 40 80 0 I 0 20 40 80
B
Figure 1: Electrophoretic analysis of duplex formation and of degradation by Sl and S7 nuclease activities. (A) Native polyacrylamide gel at 20oe of the different combinations oflabelled Dl, D2, D3, D4, D5 and D6, aps with wobble G-Tbase pair. (B) Digest ofps-D5 · D6 and ps-D3 · D4(all strands labelled) at woe with Sl nuclease. (C) Digest ofps-D5 · D6 and ps-D3 · D4 (all strands labelled) at woe with S7 nuclease.
Incubation [min)
Slructure
Enzyme SIIllnd(s)D
Gel
A
Downloaded by [New York University] at 14:25 06 December 2014
1204
8
Rippe eta/.
A
.-..
]
4
::-
6
·· ...
:i
0
.! w
2
-4
e i._. u
~
Downloaded by [New York University] at 14:25 06 December 2014
0
8 .-..
4 .......
1:2
e
2
:ie
0
_.
:i
._.
~
~
-4
-1
F 4
::-
::-
~
oi
e
e 2
u
._.
.! w
-4
220
240
260
280
Wavelength (nm)
300
220
240
260
280
~
300
Wavelength (nm)
Figure 2: Ultraviolet absorption and circular dichroism spectra of ps and aps oligonucleotides. (A-C) UV-spectra. (A) Native spectra at l0°C ofps-D5 · D6 (-), ps-Dl · D2 (---)and aps-Dl · D3 (·····).(B) Differencespectraof[ps-D5 · D6- aps-Dl · D3) at 10°C(-)andat70°C (-----)andof[ps-Dl · D2- apsDl · D3] at l0°C (---)and at70°C (·····). (C)e* in the native state at lOoC (•) and in the denatured state at 70°C (D). e* is the calculated extinction coefficientpernucleotide for a dG · dC base pair in ps-D5 · D6, including possible spectral changes (.6-edA dT) in the adjacent dA · dT residues compared to the equivalent positions in the ps-Dl · D2 sequence. This quantity was estimated by assuming additivity of unit contributions and negligible end effects, according toe* = edG·dC + 2.6-edA·dT = 25/4 · (eps-OS· 06 -21/25 ·eps01.02).As references, the spectraofpoly[d(G-C)) at l0°C (--···)and at70°C(---) in0.2mMTris-HC1,0.2 mMNaC1,0.02mMEDTA, pH 7.0, and thedifferencespectra2 · poly(d(A-C) · d(G-T)]- aps-Dl· D3 at l0°C (-----)and at 70°C (-)in 1 mM Tris-HCl, 1 mM N aCl, 0.1 mM EDTA, pH 7.0, are shown. The e260 of poly[d(A-C) · d(G-T)) was 6.5 mM (base)- 1 em_, at25oC (15). (D-F)Circulardichroism(CD)spectra. (D) Native spectra at l0°C: ps-D5 · D6(-), ps-Dl · D2 (---)and aps-Dl · D3 (·····).(E) Melted duplexes at 70°C, same symbols as before. (F) Difference spectra at lOoC: ps-D5 · D6- aps-Dl · D3 (-),ps-Dl · D2aps-Dl· D3 (---);at 70°C: ps-D5 · D6- aps-Dl· D3 (-----), ps-Dl· D2- aps-Dl· D3 (·····).
1.3
1.3
270 Wavelength [nm]
3.0
1.1
1.0
~
"'0
E-"
";"Ei
0.01
3.5
3.4
3.3
3.2
3.1
~ /"~
~
60
0.1
Na+(M)
1.0
10
10
20
30
40
50
;:§'
~
;,.;
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs a
a
Karsten Rippe , Niels B. Ramsing , Reinhard a
Klement & Thomas M. Jovin
a
a
Max Planck Institute for Biophysical Chemistry, Department of Molecular Biology , P.O. Box 2841, D-3400 , Göttingen , F.R.G. Published online: 21 May 2012.
To cite this article: Karsten Rippe , Niels B. Ramsing , Reinhard Klement & Thomas M. Jovin (1990) A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs, Journal of Biomolecular Structure and Dynamics, 7:6, 1199-1209, DOI: 10.1080/07391102.1990.10508559 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508559
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 14:25 06 December 2014
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).
A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs
Downloaded by [New York University] at 14:25 06 December 2014
Karsten Rippe, Niels B. Ramsing t, Reinhard Klement and Thomas M. Jovin * Max Planck Institute for Biophysical Chemistry Department of Molecular Biology P.O. Box 2841 D-3400 Gottingen, F.R.G. Abstract DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix ofB-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D 1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-l6°C lower and the van't Hoff enthalpy difference AllvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgC12) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-(d(T5 G~) · d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.
Introduction Pairs of deoxyoligonucleotides consisting of dA and dT sequences designed to be complementary only if the strands are oriented with the same polarity hybridize spontaneously into stable double-helical structures (1-6). Distinctive structural features ofparallel stranded DNA(ps-DNA) derived from model calculations are reverse Watson-Crick base pairing with the glycosidic bonds in a trans orientation and the lack of distinct rnajar and minor grooves. Parallel stranded duplexes exhibit spectroscopic, thermodynamic, and biochemical properties different from those of conventional antiparallel B-ONA, and are remarkably stable (1-6). For example, the enthalpy change associated with the helix-coil transition ofps-DNAis only 1020% less than that of B-ONA (5,6), in qualitative agreement with the force field calculations ofPattabiraman who originally proposed the structure (7). All parallel t Current address:
Department of Ecology and Genetics, University of Arhus, Ny Munkegade, DK-8000 Arhus C, Denmark
*Author to whom correspondence should be addressed.
1199
Rippe eta/.
1200
stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. The potential biological significance of this alternative helical structure, however, depends upon its feasibility in the case of natural sequences containing dG and dC as well. Therefore we have substituted four dA · dT base pairs of a parallel stranded linear duplex with dG · dC base pairs and compared its properties to that of the ps reference duplex consisting exclusively of dA · dT base pairs. Materials and Methods
Downloaded by [New York University] at 14:25 06 December 2014
Oligonucleotide Synthesis and Characterization
e
The oligonucleotides were synthesized, purified, labelled with 2P], and hybridized as described previously (4). The purity of the isolated end-labelled oligonucleotides was established by electrophoresis under denaturing conditions (4). No contaminating faster or slower migrating species could be detected (data not shown). Gel electrophoresis under native conditions was carried out in 14% polyacrylamide (5% crosslinking), 90 mM Tris-borate, pH 8.0, 2 mM MgC12 and at 20°C. The nuclease susceptibilities of 32P-end labelled DNAs were assessed according to Ref. 8 with Aspergillus oryzae Sl nuclease (Pharmacia) and Staphylococcus aureus (Micrococcal) nuclease S7 (Sigma). Spectroscopy
UV absorption measurements were made with a Uvikon 820 spectrophotometer equipped with computer-controlled thermostated cuvette holders and data acquisition (5) in 0.4 M NaCl and 10 mM N a-cacodylate, pH 7.1, unless otherwise indicated. The molar extinction coefficient of the oligonucleotides D 1, D2, D3 and D4 was taken to be 8.6 mM (base) -I cm- 1 at 264 nm in the denatured state (70 C standard buffer+ 0.1 M NaCl; Figure 2B) (2,4). The corresponding extinction coefficients forDS and for D6 in the denatured state were estimated bycombiningthe dA · dTvaluecited above for D 1-4 with the extinction coefficient for poly [d(G-C)] in the native (9) and denatured (Ramsing &Jovin, in preparation) state, and the corresponding value for denatured poly[dC] (10). We calculated ane264 of8.6and8.4mmol base- 1 em-I forDS and D6, respectively, at 70°C. Circular dichroism (CD) spectra were acquired with a Jobin Yvon Model V Dichrograph in 2 mM MgC12, 10 mM Na-cacodylate, pH 7.1. Model Force Field Calculations
Energy minimization was with the AMBER-PMF program ( 11) using 0.1 M N aCl as the supporting electrolyte in conjunction with the INSIGHT (Biosym Tech.) molecular modelling package and a PS390 Evans and Sutherland graphics workstation. Results and Discussion Design of Oligonucleotides
The set of25-nt oligonucleotides D 1, D2, D3 and D4 shown below were designed by
1201
Parallel Stranded Linear DNA
Downloaded by [New York University] at 14:25 06 December 2014
a computer aided search for complementary sequences capable of forming parallel stranded (ps) helices with minimal interference from competitive antiparallel (aps) secondary structures (4). The two ps-DNA duplexes, ps-D 1 · D2, ps-D3 · D4, and the aps-Dl · D3 and aps-D2 · D4 references, were extensively characterized with respect to spectroscopic and thermodynamic properties (4-6) and were also tested as substrates for several DNA processing enzymes and chemical reagents (8). A comparison of the two combinations ps-D 1 · D2 and ps-D3 · D4 with the corresponding aps molecules revealed the same characteristic differences found previously with other ps-DNA constructs (1-6). In order to determine the influence of dG and dC residues on the properties of the parallel stranded helix, we synthesized two other 25-nt oligonucleotides designated as D5 and D6, in which four dA(D5) or four dT (D6) bases were replaced by dG and dC, respectively. Synthesis and purification were according to Ref. 4. The following combinations of the Dl-D6 oligonucleotides were stable (see below): PanlleliiDDcled duplexes
Anlipanllelltranded duplexes
01: 5 1 -AAAAAAAAAATAATTTTAAATATTT-3 1 5 I -TTTTTTTTTTATTAAAATTTATAAA-3 I :02 1
03: 5 -AAATATTTAAAATTATTTTTTTTTT-3
.............•••.........
1
5 I -TTTATAAATTTTAATAAAAAAAAAA-3 I :04 OS: 5 1 -AAAAAQAAAQTAQTTTTAAQTATTT-3 1 •••••••••a••••••••••••••• 5 I -TTTTTCTTTCATCAAAATTCATAAA-3 I :06
01: 5 1 -AAAAAAAAAATAATTTTAAATATTT-3 1 3 I -TTTTTTTTTTATTAAAATTTATAAA-5 I :03
........................ .
D2: 5 I -TTTTTTTTTTATTAAAATTTATAAA-3 I 3 I -AAAAAAAAAATAATTTTAAATATTT-5 I :04 ~: 5 1 -AAAAAQAAAGTAQTTTTAAQTATTT-3 1
••••.•...•..•......•.....
3 I -TTTTnTTT'fAT'fAAAATT'fATAAA-5 1 :03
Electrophoretic Properties The generation and stability of the proposed duplex structures were assessed by gel electrophoresis under native conditions (2 mM MgC1 2, 20°C; Figure lA). The only duplexes formed in addition to those described previously [ps-Dl · D2, ps-D3 · D4, aps-Dl · D3, aps-D2 · D4 (4,5)] corresponded to the combination D5+ D6 (designated ps-D5 · D6) that has a sequence with four dG · dC base pairs and complementarity in a parallel orientation, and D5+ D3 (antiparallel with four dG · dT mismatches). The other potential ps combinations involved either opposing dG and dT (D5+ D2) or dA and dC (Dl + D6) migrated in the position of single stranded species (with Dl + D6 showing a slightly lower mobility).
Spectroscopic Properties The ultraviolet absorption and circular dichroism spectra of the ps-D5 · D6 duplex are indicative of a base-paired structure resembling that of ps duplexes consisting onlyofdA · dTbasepairs. Thus, both ps-D5 · D6andps-Dl · D2 show a characteristic blue shift in the native UV spectra relative to aps-D 1 · D3 (Figure 2A), a feature particularly evident in the difference spectra (Figure 2B) and attributable to the dA · dT part of the sequence. Upon denaturation, the spectra (Figure 2B) demonstrate only the differences expected from the base composition. In Figure 2C the specific
Rippe eta/.
1202
Downloaded by [New York University] at 14:25 06 December 2014
dG · dC contribution in ps-05 · 06 was calculated by subtracting the fractional dA · dT absorbance estimated from the spectra of ps-01 · 02. In the denatured state, the specific dG · dC absorbance is similar to that of poly[d(G-C)] and to the estimated dG · dC contribution of poly d[(A-C) · d(G-T)]. In the native state, however, the calculated dG · dC spectrum ofps-05 · 06 exhibits a higher absorbance, a general broadening, and a red shift of the peak compared to the reference ONAs. [As noted in the legend to Figure 2C, perturbations of the adjoining dA · dT base pairs may also contribute.] TheCOspectrumofps-05 · 06comparedtothatoftheaps-Ol· 03referenceinthe native state (Figure 20) shows the same features observed with ps-01 · 02 above 265 nm (seen as a difference spectrum in Figure 2F) whereas at 250 nm the dichroism is reduced. The spectra in the denatured state are nearly similar for all three combinations (Figure 2E) showing only slight deviations attributable to the different base composition. Helix-Coil Transition
Salt-dependent thermal transitions of various duplexes were monitored by the increase of UV absorption upon melting and represented as three-dimensional plots (Figure 3A-E). The absorbances at each temperature T and wavelength were normalized to the values at the initial (low) temperature T0 in order to reveal the characteristic differences between the ps and aps conformation (2,4,5,6). The combination ps-05 · 06 in MgC12 or NaCl displays the same distinctive hyperchromicity pattern found with ps-01 · 02, ps-03 · 04 and otherps ONAs (2,4,5,6). The melting temperature Tm of ps-05 · 06 is 10-16°C lower than that of the corresponding duplex ps-01 · 02 composed only of dA · dT base pairs and has the same salt dependence as the reference ONAs (Figure 3F, Table I). The van't Hoff enthalpy difference tll/vH for the helix-coil transition of ps-05 · 06 is reduced by 20% (in NaCl) and 10% (in MgC12) compared to that ofps-01 · 02, although it is greater than the value determined for a 21-nt ps duplex consisting exclusively of dA · dT base pairs (2,5,6). We calculated the mean dG · dC contribution per mole of nearest neighbors (N.N.) ofps-05 · 06, ~hdG·dO by assuming that the substitution of the 4 dG · dC base pairs outof25leads to a change in 8 N.N. interactions. The resulting expression (~hdG. d~ = [tll/ps-os. 06 - 16/24 · tll/ps-Ot. 02 )18) yields 8 kJ ·mol-t in NaCl and 14 kJ · mol- in MgC12, respectively, and can be compared to the mean values for the
Legend for Color Folio Figure 4: Model of dG · dC base pair in ps(d(TsGA5) · d(A5CT5)] optimized by force field calculations. (A) Side-view of the central three base pairs; 5' ends are at the bottom. (B) eros-sectional view of the central dG · dC (ball and stick) and underlying dA · dT (space filling) base pair. Color coding: sugars, white; phosphates (including 03' and 05'), red; adenine, cyan; thymine, yellow; thymine methyl groups, turquoise; guanine, blue; cytosine, green. The figure was generated by the SCHAKAL v86b program of Dr. E. Keller, Univ. of Freiburg, FRG. The model was generated by extracting the 3' terminalll residues from an optimized 21-nt ps duplex (2) and refining the resultant 11-nt ps-(d(TsAt,) · d(A5T 6)] duplex as well as the sequence in which the central dA · dT base pair was replaced by dG · dC.
Downloaded by [New York University] at 14:25 06 December 2014
Downloaded by [New York University] at 14:25 06 December 2014
I ss
1,2 1,3 1,4 1,5 1,6 2 ps aps - ss
2,3 2,4 2,5 2,6 3 - aps - ss
3,4 3,5 3,6 4 ps - ss
4,5 4,6 - ss
5,6 6 ps ss
- - - - - - - - - - - - - native------------denaturing
- - - - - denaluring - - - - -
c
- - - - - S I nuclease---- - - - Micrococcus nMclease - - - - - 5,6-3,4-- - - - 5,6-- - - - 3,4-- - ps (G·C)- - - - ps - - - - - ps (G·C)- - - - ps - - 0 I 0 20 40 80 0 I 0 20 40 80 0 I 0 20 40 80 0 I 0 20 40 80
B
Figure 1: Electrophoretic analysis of duplex formation and of degradation by Sl and S7 nuclease activities. (A) Native polyacrylamide gel at 20oe of the different combinations oflabelled Dl, D2, D3, D4, D5 and D6, aps with wobble G-Tbase pair. (B) Digest ofps-D5 · D6 and ps-D3 · D4(all strands labelled) at woe with Sl nuclease. (C) Digest ofps-D5 · D6 and ps-D3 · D4 (all strands labelled) at woe with S7 nuclease.
Incubation [min)
Slructure
Enzyme SIIllnd(s)D
Gel
A
Downloaded by [New York University] at 14:25 06 December 2014
1204
8
Rippe eta/.
A
.-..
]
4
::-
6
·· ...
:i
0
.! w
2
-4
e i._. u
~
Downloaded by [New York University] at 14:25 06 December 2014
0
8 .-..
4 .......
1:2
e
2
:ie
0
_.
:i
._.
~
~
-4
-1
F 4
::-
::-
~
oi
e
e 2
u
._.
.! w
-4
220
240
260
280
Wavelength (nm)
300
220
240
260
280
~
300
Wavelength (nm)
Figure 2: Ultraviolet absorption and circular dichroism spectra of ps and aps oligonucleotides. (A-C) UV-spectra. (A) Native spectra at l0°C ofps-D5 · D6 (-), ps-Dl · D2 (---)and aps-Dl · D3 (·····).(B) Differencespectraof[ps-D5 · D6- aps-Dl · D3) at 10°C(-)andat70°C (-----)andof[ps-Dl · D2- apsDl · D3] at l0°C (---)and at70°C (·····). (C)e* in the native state at lOoC (•) and in the denatured state at 70°C (D). e* is the calculated extinction coefficientpernucleotide for a dG · dC base pair in ps-D5 · D6, including possible spectral changes (.6-edA dT) in the adjacent dA · dT residues compared to the equivalent positions in the ps-Dl · D2 sequence. This quantity was estimated by assuming additivity of unit contributions and negligible end effects, according toe* = edG·dC + 2.6-edA·dT = 25/4 · (eps-OS· 06 -21/25 ·eps01.02).As references, the spectraofpoly[d(G-C)) at l0°C (--···)and at70°C(---) in0.2mMTris-HC1,0.2 mMNaC1,0.02mMEDTA, pH 7.0, and thedifferencespectra2 · poly(d(A-C) · d(G-T)]- aps-Dl· D3 at l0°C (-----)and at 70°C (-)in 1 mM Tris-HCl, 1 mM N aCl, 0.1 mM EDTA, pH 7.0, are shown. The e260 of poly[d(A-C) · d(G-T)) was 6.5 mM (base)- 1 em_, at25oC (15). (D-F)Circulardichroism(CD)spectra. (D) Native spectra at l0°C: ps-D5 · D6(-), ps-Dl · D2 (---)and aps-Dl · D3 (·····).(E) Melted duplexes at 70°C, same symbols as before. (F) Difference spectra at lOoC: ps-D5 · D6- aps-Dl · D3 (-),ps-Dl · D2aps-Dl· D3 (---);at 70°C: ps-D5 · D6- aps-Dl· D3 (-----), ps-Dl· D2- aps-Dl· D3 (·····).
1.3
1.3
270 Wavelength [nm]
3.0
1.1
1.0
~
"'0
E-"
";"Ei
0.01
3.5
3.4
3.3
3.2
3.1
~ /"~
~
60
0.1
Na+(M)
1.0
10
10
20
30
40
50
;:§'
~
;,.;
