Solution Structure of a DNA Complex with the Fluorescent Bis

Bioconjugate Chem. 1998, 9, 331−340

331

Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Modified on the Benzothiazole Ring Michael Petersen and Jens Peter Jacobsen* Department of Chemistry, Odense University, Odense M, DK-5230 Denmark. Received July 21, 1997; Revised Manuscript Received December 3, 1997

We have used two-dimensional 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5′-CGCTAGCG-3′)2 complexed with the bis-intercalating dye 1,1′-(4,4,8,8-tetramethyl4,8-diazaundecamethylene)bis-4-[3-ethyl-2,3-dihydro(benzo-1,3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTOEt). The determination of the structure was based on a total relaxation matrix analysis of the NOESY cross-peak intensities. DQF-COSY spectra were used to obtain coupling constants for the deoxyribose ring protons. The coupling constants were transformed into angle estimates. The NOE-derived distance and dihedral restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the TOTOEt complex from both A-form and B-form double-stranded (ds) DNA starting structures, giving a total of 40 final structures. Since many NOE contacts were observed between TOTOEt and dsDNA, the resulting structure has a fairly high resolution and allows determination of local features in the dsDNA structure after TOTOEt binding. The root-mean-square (rms) deviation of the coordinates for the 40 structures of the complex was 0.52 Å. The local DNA structure is distorted in the complex. The helix is unwound by 80° and has an overall helical repeat of 12 base pairs, caused by bis-intercalation of TOTOEt. The benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTOEt molecule. The site selectivity of TOTOEt for the CTAG‚CTAG site is explained by its ability to adapt to the base pair propeller twist of dsDNA to optimize stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. The polypropylene amine linker chain is located in the minor groove of dsDNA. The N-ethyl group on the benzothiazole of TOTOEt is placed in the major groove pointing toward the center of the oligonucleotide and the dyad symmetry axis of the complex. This orientation seems to make it feasible to create a TOTO analogue with a linker connecting the two chromophores in the major groove. The design of such an analogue and a macrocyclic analogue with a linker in both the major groove and the minor groove seems to be straightforward.

INTRODUCTION

Scheme 1. TOTO and TOTOEt Numbering Scheme

Progress in cancer research indicates that neoplasia is a malady of genes (Bishop, 1987). Although several potent antitumor drugs do not interact with nucleic acids, there is accumulating evidence that suggests that the malignant transformation of cells is due to alterations of particular genes. Thus, the development and investigation of DNA-interacting substances is called for. A prominent class of DNA-targeting drugs are intercalators and bis-intercalators (e.g. amsacrine, daunomycin, and * To whom correspondence should be addressed. E-mail: [email protected]. 1 Abbreviations: AMBER, assisted model building with energy refinement; DNA, deoxyribose nucleic acid; DMSO, dimethyl sulfoxide; DQF-COSY, double-quantum-filtered correlation spectroscopy; dsDNA, double-stranded DNA; DSS, 2,2-dimethyl-2silapentane-5-sulfonate; EDTA, ethylenediaminetetraacetic acid; HSQC, heteronuclear single-quantum coherence; MARDIGRAS, matrix analysis of relaxation for discerning the geometry of an aqueous structure; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; RANDMARDI, randomized MARDIGRAS; RMD, restrained molecular dynamics; TOCSY, total correlation spectroscopy; TOTO, 1,1′-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis-4-[3-methyl-2,3-dihydro(benzo-1,3-thiazolyl)-2methylidene]quinolinium tetraiodide; TOTOEt, 1,1′-(4,4,8,8tetramethyl-4,8-diazaundecamethylene)bis-4-[3-ethyl-2,3-dihydro(benzo-1,3-thiazolyl)-2-methylidene]quinolinium tetraiodide; TPPI, time-proportional phase incrementation.

adriamycin). To gain knowledge of the often subtle features that govern drug-DNA recognition, the need for high-resolution structures of drug-DNA complexes is imperative. Recent research in our laboratory has focused on the bis-intercalating drug TOTO1 (Scheme 1). We have examined both the sequence specificity and the kinetics

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332 Bioconjugate Chem., Vol. 9, No. 3, 1998

of association and dissociation for dsDNA complexes with both TOTO and a number of TOTO analogues (Faridi et al., 1997; Hansen et al., 1996; Jacobsen et al., 1995; Spielmann et al., 1995b; Stærk et al., 1997). TOTO bisintercalates into dsDNA by nearest neighbor intercalation and shows sequence selectivity toward the (5′-CTAG3′)2 site (Hansen et al., 1996; Jacobsen et al., 1995). The structure of the complex implies that modifications on the benzothiazole nitrogen would be expected to be positioned in the major groove on complex formation with dsDNA (Spielmann et al., 1995b). However, the binding of TOTO has been shown to be very sensitive to even small modifications on the oligonucleotide (Hansen et al., 1996). Consequently, there is a risk that slight changes of the chromophore of TOTO would destroy the binding affinity and selectivity of the molecule. Our long-term objective is to design a TOTO analogue with a linker connecting the two chromophores in the major groove, subsequently with a linker in both the major groove and the minor groove. As a primary modification, the N-methyl group of TOTO was substituted with a N-ethyl group to give the compound 1,1′(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis-4-[3ethyl-2,3-dihydro(benzo-1,3-thiazolyl)-2-methylidene]quinolinium tetraiodide (TOTOEt, Scheme 1). We have used 1H NMR spectroscopy to determine the solution structure of the DNA oligonucleotide d(5′-CGCTAGCG3′)2 complexed with the bis-intercalating TOTOEt. The determination of the structure was based on the protocol described by James and co-workers (Schmitz and James, 1995) combined with inclusion of dihedral angle estimates obtained from coupling constants in the deoxyribose ring protons. This study was carried out to investigate whether this modification implied a dramatic change of the binding and selectivity of the molecule. Furthermore, this structural study was aimed at determining the position of the N-ethyl group in a dsDNA complex and the feasibility of substituting larger groups than ethyl, eventually a linker, at the benzothiazole nitrogen while still maintaining proper affinity and selectivity with respect to dsDNA. EXPERIMENTAL PROCEDURES

Sample Preparation. TOTOEt was a gift from E. B. Pedersen (Department of Chemistry, Odense University, Denmark). The purified oligonucleotide d(CGCTAGCG)2 was purchased from DNA technology (Århus, Denmark) and used without further purification. The numbering scheme for the double-stranded oligonucleotide is

TOTOEt is almost insoluble in water, and the complexes with dsDNA were therefore made with the procedure described earlier (Jacobsen et al., 1995). The NMR samples were prepared by dissolving the complexes in 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0), 0.05 mM Na EDTA, 0.01 mM NaN3, and 0.1 mM DSS. For experiments carried out in D2O, the solid complex, lyophilized three times from D2O, was redissolved in 99.96% D2O (Cambridge Isotope Laboratories). A mixture of 90% H2O and 10% D2O (0.5 mL) was used for experiments examining exchangeable protons. The sample was kept in a NMR tube under nitrogen. The final concentration of the complex was 2 mM. NMR Experiments. NMR experiments were performed on a Varian Unity 500 spectrometer at 27 °C

Petersen and Jacobsen

unless otherwise stated. NOESY spectra of the complex were acquired in D2O using 1024 complex points in t2 and a spectral width of 5000 Hz. A total of 512 t1 experiments were recorded using the States phase cycling scheme. The residual signal from HOD was removed by presaturation. NOE buildups were obtained by recording NOESY spectra with mixing times of 50, 100, 150, and 200 ms with 64 scans acquired for each t1 value and a repetition delay of 2.8 s between each scan. The NOESY spectra were obtained sequentially without removing the sample from the magnet. TOCSY spectra of the complex with mixing times of 30 and 90 ms were obtained in the TPPI mode using 1024 complex points in t2 and 512 t1 experiments with 64 scans each. DQF-COSY spectra were recorded using the States phase cycling scheme with 1024 complex points in t2 and 1024 t1 experiments with 64 scans each. Removal of the residual HOD signal was in either case carrieed out with presaturation. A NOESY spectrum of the complex in H2O with a mixing time of 160 ms was obtained by using a NOESY pulse sequence where the last 90° pulse is replaced by a 1-1 jump-return pulse to suppress the solvent signal (Gue´ron and Plateau, 1982). A Watergate NOESY spectrum in H2O with a mixing time of 50 ms was recorded on a Varian Unity+ 500 spectrometer. Both NOESY spectra in H2O were acquired with a spectral width of 10 000 Hz in 2048 complex points using 512 t1 experiments with 64 scans acquired in each t1 experiment. A 1H, 13C HSQC spectrum in D2O was recorded on a Varian Unity+ 500 spectrometer. A NOESY spectrum with mixing time of 500 ms was recorded of the free TOTOEt in DMSO-d6. The acquired data were processed using FELIX (version 2.35, Biosym/MSI, San Diego, CA). All spectra were apodized by a skewed sine bell squared function in both dimensions. The four spectra used in the buildup study were zero-filled to 2K points to enhance digital resolution and baseline corrected by first-order standard polynomial fits in both dimensions. The NOESY spectrum recorded in H2O was baseline corrected by a third-order standard polynomial in F2 and seventh-order one in F1. The DQFCOSY spectrum was zero-filled to 4K points. The spectra were assigned by conventional methods (Wu¨thrich, 1986; Feigon et al., 1983; Hare et al., 1983; Scheek et al., 1983, 1984; Boelens et al., 1985). Distance and Angle Restraints. The structure determination was performed using the protocol described by James and co-workers (Schmitz and James, 1995) combined with inclusion of dihedral angle estimates obtained from coupling constants in the deoxyribose ring protons in the following way. The upper and lower diagonal parts of each of the four NOESY buildup spectra were integrated separately with FELIX, yielding a total of eight peak intensity sets. The RANDMARDI procedure (Liu et al., 1995) of the complete relaxation matrix analysis method MARDIGRAS (Borgias et al., 1990; Borgias and James, 1990) was used to calculate interproton distance RMD bounds from the resulting integrated peak intensities. In the calculations, a noise level of 5 times the integrated intensity of the smallest cross-peak was used as the “noise” range. The dynamic range of observed cross-peak intensities was 2000. In the RANDMARDI procedure, 30 different intensity sets from each experimental data set were generated, and MARDIGRAS calculations were performed on all of them. Resulting distances were then averaged together to form one bounds file for each of the eight data sets. The bounds files from all of the mixing times were then combined into a single bounds file from which the RMD restraint file was generated. Upper and lower bounds

DNA Complex with TOTO

Bioconjugate Chem., Vol. 9, No. 3, 1998 333

were average interproton distances ( one standard deviation calculated from all of the MARDIGRAS runs. An additional 22 distance restraints were included to mimic Watson-Crick hydrogen bonding throughout the calculations. Three hydrogen bonds were included for each of the six G‚C base pairs and two hydrogen bonds for each of the A‚T base pairs with lower and upper bounds of 1.7 and 2.1 Å, respectively. The H1′ to H2′ and H2′′ region of the DQF-COSY spectrum was used as input to CHEOPS to obtain J coupling information for the H1′, H2′, H2′′, and H3′ deoxyribose ring protons (Gochin et al., 1990; Macaya et al., 1992). The ring conformations were analyzed using the PSEUROT procedure (de Leeuw and Altona, 1983). Restrained Molecular Dynamics. TOTOEt was manually docked into the model of either A-form or B-form DNA in INSIGHTII (version 95.0, Biosym/MSI) to obtain the starting models for the structure refinements. The TOTOEt molecule was built using INSIGHTII. The potentials for both DNA and TOTOEt were assigned by the AMBER force field, modified to accommodate the benzothiazole sulfur atom of TOTOEt. Partial charges for TOTOEt were calculated using ab initio GAUSSIAN94 calculations. The distance restraints obtained from RANDMARDI and dihedral angle restraints from PSEUROT were incorporated into energy minimization and an RMD procedure using DISCOVER (version 95.0, Biosym/MSI). An initial energy minimization was followed by 28 ps of restrained molecular dynamics, at 600 K for 4 ps, followed by cooling to 200 K in 50 K steps of 3 ps each. The final structure was then energy minimized to a maximum derivative of 0.01 Å. The pseudo-energy terms used to enforce the restraints were

{ {

k1(r - r1)2 ENOE ) 0 k1(r - r2)2 k2(θ - θ1)2 Edihedral ) 0 k2(θ - θ2)2

when r1 > r when r2 g r g r1 when r > r2 when θ1 > θ when θ2 g θ g θ1 when θ > θ2

where r1 and r2 are the lower and upper distance bounds determined from the cross-peak volumes, respectively, k1 and k2 are the force constants, and θ1 and θ2 are the lower and upper dihedral angle bounds determined from the DQF-COSY spectral simulations, respectively. For NOEderived distance restraints a force constant k1 of 50 kcal/ (mol Å2) was employed and for dihedral angle restraints a force constant k2 of 25 kcal/(mol Å2). The width of the flat region of the potential well reflects the accuracy of a given constraint. A distance-dependent dielectric ( ) r) was employed to account for solvent effects. Helix parameters were calculated with the program CURVES 5.1 (Lavery and Sklenar, 1988, 1989). RESULTS

Spectral Analysis. The 1D spectrum of the dsDNATOTOEt complex exhibits sharp lines, with line widths as small as those observed for the dsDNA-TOTO complex. There is no sign whatsoever of any minor forms. We therefore conclude that TOTOEt has a strong preference for the (5′-CTAG-3′)2 site. Observation of lines for just one dsDNA strand and one thiazole orange chromophore shows that the dyad symmetry of the d(CGC-

a

b

Figure 1. (a) H1′-aromatic part of the 150 ms NOESY spectrum of the TOTOEt complex. The sequential H1′-H6/H8H1′ connectivity pathway is indicated by a solid line. The interrupted connectivities at the 5′-C3pT4-3′ and 5′-A5pG6-3′ base pair steps due to intercalation sites are indicated by dotted lines. It is also possible to follow the H3′-H6/H8-H3′ connectivity pathway with similar interrupted connectivities. A few of the observed cross-peaks between TOTOEt and the oligonucleotide are also marked. It is noteworthy that the cross-peak between TOTOEt H15 and A5 H1′ has a measured volume which is approximately 100 times smaller than the observed cross-peak between TOTOEt H8 and H16. This demonstrates the large dynamic range and precision of the measured volumes that can be obtained from the spectra. (b) Methyl-aromatic part of the 160 ms NOESY spectrum recorded in H2O. The assignments of the C3 and A5 amino protons, the thymidine methyl protons, and some of the TOTOEt protons are indicated. Crosspeaks between TOTOEt CH3(6) and the amino protons of C3 and A5 unambiguously place the benzothiazole N-ethyl group in the major groove of the dsDNA.

TAGCG)2 oligonucleotide is retained on complex formation with TOTOEt. The NOESY spectrum of this complex exhibits the characteristic features of dsDNA sequential connectivities. However, the sequential pathways are interrupted at the 5′-C3pT4-3′ and 5′-A5pG6-3′ base pair steps. This is clear evidence of bis-intercalation (Hansen et al., 1996; Jacobsen et al., 1995; Spielmann et al., 1995b). Parts of the NOESY spectra are shown in Figure 1. The NOESY spectra of the complex also contain a multitude of intermolecular cross-peaks between dsDNA and TOTOET as well as intramolecular TOTOEt cross-peaks. These were assigned in a similar way as previously



Table 1. Chemical Shift Values of the TOTOEt Complex with d(CGCTAGCG)2 at 27 °Ca H8-H6 H5-CH3-H2 H1′ H2′ H2′′ H3′ H4′ H5′ H5′′ H1-H3 H4-H6 H4-H6

a

C1

G2

C3

T4

A5

G6

C7

G8

7.55 (7.65) 5.80 (5.92) 5.68 (5.81) 1.91 (2.00) 2.31 (2.45) 4.63 (4.76) 4.01 (4.09) 3.67 (3.74) 3.67 (3.74)

7.76 (7.99)

6.48 (7.44) 4.62 (5.39) 5.97 (5.98) 1.82 (2.07) 2.45 (2.51) 4.62 (4.78) 4.05 (4.24) 4.05 (4.18) 4.16 (4.26)

7.08 (7.41) 0.82 (1.70) 4.96 (5.60) 1.92 (2.14) 1.99 (2.44) 4.65 (4.88) 4.15 (4.14) 4.15 (4.09) 4.10 (4.09) 13.57 (13.76)

8.20 (8.23) 7.03 (7.37) 5.54 (6.02) 2.87 (2.74) 2.75 (2.88) 5.12 (5.05) 4.32 (4.40) 3.95 (4.15) 4.12 (4.06)

7.83 (7.70)

7.32 (7.30) 5.33 (5.34) 5.77 (5.76) 1.88 (1.87) 2.32 (2.33) 4.80 (4.79) 4.20 (4.14) 4.20 (4.10) 4.14 (4.19)

7.91 (7.93)

-

5.72 (5.97) 2.40 (2.71) 2.40 (2.77) 4.84 (5.00) 4.25 (4.38) 3.90 (4.11) 4.02 (4.01) 12.78 (13.02)

8.03 (8.27) 6.54 (6.58)

5.82 (5.71) 2.68 (2.50) 2.68 (2.60) 4.88 (4.95) 4.57 (4.37) 4.02 (4.19) 4.39 (4.19) 12.14 (12.76)

7.22 (7.81) 6.71 (6.54)

6.12 (6.16) 2.60 (2.60) 2.35 (2.37) 4.66 (4.66) 4.07 (4.16) 4.07 (4.06) 4.17 (4.06) -

8.19 (8.34) 6.43 (6.47)

TOTOEt

shift

TOTOEt

shift

TOTOEt

shift

TOTOEt

shift

H1 H2 H3 H4 CH3(6) CH2(6)

7.59 7.48 7.58 7.46 1.30 4.18

H8 H9 H10 H13 H14 H15

6.16 6.50 7.97 6.99 6.97 6.69

H16 H19′ H19′′ H20′ H20′′ H21′

7.42 4.13 4.62 2.49 2.57 3.65

H21′′ CH3(22) H23′ H23′′ H24

3.73 3.31 3.48 3.48 2.49

Compared to free dsDNA chemical shifts given in parentheses. The values are given relative to DSS.

published (Jacobsen et al., 1995; Spielmann et al., 1995b). The DQF-COSY spectrum was used for the assignment of proton resonances in the linker of TOTOEt. The combined use of DQF-COSY spectra and NOESY spectra with short mixing times allows unambiguous assignments of the H2′ and H2′′ resonances. The HSQC spectrum was used to completely establish the assignments of the H3′ and H4′ protons. The chemical shift values of the protons in the complex are given in Table 1. The NOESY spectra of the complex recorded in H2O contain the normal Watson-Crick NOE connectivity pattern justifying the inclusion of 22 hydrogen bond distance restraints. Chemical shift values of labile protons are included in Table 1. Furthermore, the spectra in H2O contain distinct cross-peaks between the thymidine imino proton (T4-H3) and the amino protons of the base-paired adenosine (A13-H6). These peaks are absent in normal dsDNA at room temperature due to exchange. The lowering of the exchange rates of the adenosine amino protons on complex formation is a consequence of the bis-intercalation. Similarly, exchange cross-peaks between water and the thymidine imino protons are absent at room temperature, and only very small ones are observed for the G6 and G14 imino protons. The G2 and G10 imino protons yield an exchange cross-peak to water of normal intensity. Studies of the conformation of the free TOTOEt in DMSO-d6 using NOESY and DQF-COSY spectra reveal that the relative conformation of the two ring systems in each chromophore is close to that indicated in Scheme 1. This is a consequence of a prominent NOESY crosspeak between H8 and H16. We have found an identical conformation of the aromatic ring systems in the dsDNA-TOTOEt complex. The relative conformation of the aromatic ring systems about the cyanine methine bonds in the chromophores is therefore not affected by intercalation into dsDNA. Cross-peaks in the NOESY spectra unambiguously show that the poly(propylene amine) linker is positioned in the minor groove. This is demonstrated by a large number of cross-peaks. The H2 of the adenines gives cross-peaks to the linker protons H21, H22, and H23 and the H1′ proton of G6 to the linker H19, H21, and H22 protons, and the H4′ of the adenines and the guanines

Table 2. Coupling Constants for the Sugar Protons in the dsDNA-TOTOEt Complexa C1 G2f C3 T4 A5 G6f C7 G8

J1′2′

J1′2′′

J2′2′′b

corrc

%Sd

PSe

8.0 6.0 9.6 10.9 7.8 7.8

6.2 7.0 4.5 4.4 6.4 5.7

-15.0 -15.0 -15.0 -15.0 -15.0 -15.0

0.96 0.96 0.95 0.93 0.95 0.96

88 g 84 87 92 78

164.9 134.0 154.2 119.4 173.7

a Derived from the H1′-H2′ and H1′-H2′′ cross-peaks in the DQF-COSY spectrum using CHEOPS. b J2′2′′ coupling constants were kept fixed at -15.0 Hz. c Correlation coefficient (Macaya et al., 1992). d Percent S-type sugar conformation calculated using PSEUROT. e Pseudorotation angle calculated for the S-type sugar using PSEUROT. The puckering amplitude was kept constant at 36°. f G2 and G6’s sugar puckers could not be analyzed due to spectral overlap in the DQF-COSY spectrum. g No minimum could be found for the sugar pucker parameters for this nucleotide; see the following discussion.

in the binding site also shows cross-peaks to linker protons. Cross-peaks between TOTOEt CH3(6) and CH2(6) and the amino protons of C3 and A5 unambiguously place the benzothiazole N-ethyl group in the major groove of the dsDNA. The relative intensities of these cross-peaks in the NOESY spectrum recorded in H2O with a short mixing time (50 ms) established that the N-ethyl group is pointing toward the center of the oligonucleotide. Deoxyribose Ring Conformations. Due to the dyad symmetry of the TOTOEt-d(CGCTAGCG)2 complex, only eight spectroscopically distinct deoxyribose residues are observed in the DQF-COSY spectrum. Two of these, G2 and G6, are hampered by severe overlap of the H2′ and H2′′ resonances, making use of cross-peaks from these residues impossible. For the remaining six residues, CHEOPS simulations of the DQF-COSY spectrum returned values that correlated well (>0.93) with the experimental spectrum (Macaya et al., 1992). The J coupling constants returned by CHEOPS were used as input for PSEUROT. The PSEUROT minimization converged for five of the six residues analyzed by CHEOPS, producing the values given in Table 2. Structure Calculations. More than 450 NOE crosspeaks were observed in the NOESY spectrum with a



Table 3. Covalently Determined Interproton Distances (Å) in TOTOEt and a Comparison between Actual Values and Values Obtained from MARDIGRAS Calculations atom

pair

actual valuea

lower boundb

upper boundb

widthb

violation

CH3(6) H9 H10 H14 H15

CH2(6) H10 H13 H16 H16

2.36 2.36 4.66 4.22 2.41

2.00 2.12 3.55 3.57 2.23

2.12 2.29 5.25 5.69 2.38

0.12 0.17 1.70 2.12 0.15

0.24 0.07

a

0.03

b

Obtained from GAUSSIAN94 calculations. Obtained from MARDIGRAS calculations and derived from the average distance ( one standard deviation. Table 4. Number of Distance Restraints Used in the RMD Calculations of the TOTOEt Complexa intraresidue intra- basesugar sugar total C1

3

5

8

G2

8

5

13

C3

3

2

5

T4

2

6

8

16 11 2 4 49

7 6 7 4 42

23 17 9 8 91

A5 G6 C7 G8 total

interresidue

dye-DNA

dye-dye

C1-G2 6b

C3-TOTOEt 19 5 (7) G2-C3 3 T4-TOTOEt 7 (2b) C3-T4 2 A5-TOTOEt 18 (3) T4-A5 12 G6-TOTOEt (2) 3 (0) A5-G6 2 G6-C7 6 C7-G8 5b total 38

total 45

total 19

a

The restraints were derived from MARDIGRAS calculations unless otherwise stated. Restraints derived from the NOESY spectrum recorded in H2O are tabulated in parentheses. The 193 NOE connectivities gave a total of 386 symmetry-related distance restraints; 22 distance restraints ensuring Watson-Crick base pairing were also included in the RMD calculations. b One of the restraints was derived directly from inspection of the spectra and used with loose bounds.

mixing time of 200 ms. Some of these resulted predominantly from spin diffusion and were consequently not observed in NOESY spectra with shorter mixing times. The accuracy of the integration of some cross-peaks was hampered by spectral overlap. These cross-peaks were therefore not included in the MARDIGRAS calculations. The integrated intensities from a total number of 3056 in eight sets of 382 NOESY cross-peak intensities were used in the total relaxation matrix analysis. Of these, 2880 cross-peak intensities (360 per set) corresponded to interproton distances that are not covalently fixed in either the dye or the dsDNA in the complex. Cross-peak integrals that corresponded to fixed distances in the dsDNA were not converted into distance restraints for use in the RMD simulations. The RANDMARDI calculations returned 362 interproton distances based on the eight sets of 382 NOE cross-peak intensities. These were distributed as given in Table 4. MARDIGRAS uses internal calibration when it iteratively fits the data to a model of the molecule. In particular, fixed distances were used to normalize the other distances. These fixed distances include the H5H6 distance in cytosine, the 5-methyl-H6 distance in thymidine, and the H2′-H2′′ distance in deoxyribose. NOE intensities for these known distances are used to normalize the NOE intensities in the model. The program does not reset covalently determined proton-proton distances within TOTOEt because these distances intentionally were left out of the reference file. The magnetization transfer for these protons is calculated and

provides a check of how well the RANDMARDI procedure is working. Good agreement between the actual distances of the fixed bonds and the experimentally determined distances indicates that the RANDMARDI procedure yields accurate interproton distances (Table 3). The MARDIGRAS calculations were performed with various values of the correlation time for the complex in the range of 2-5 ns. The results were fairly insensitive to the actual value, and a correlation time of 3.75 ns was chosen for the final calculation. No symmetry was enforced during the RANDMARDI procedure. The bounds for symmetry-related restraints were calculated by averaging the symmetry-related upper and lower bounds. An additional 0.2 Å was added to all upper bounds of the distance restraints returned by RANDMARDI for use in the RMD calculations to circumvent any problem that may occur due to an incomplete description of the relaxation matrix (cf. Table 3). No additional symmetry was enforced during the RMD calculations. The range of differences between the upper and lower bounds for the 362 NOE-derived distance restraints used was between 0.27 and 3.13 Å with an average flat well potential width of 1.19 Å. This range between the upper and lower bounds represents the accuracy of the restraints calculated from the NOESY data. Weak NOESY cross-peaks that arose primarily from spin diffusion had the greatest difference between their upper and lower bounds. Interproton distances involving exchangeable DNA amino and imino protons were derived from the NOESY spectrum of the complex recorded in H2O with a mixing time of 160 ms. The “jump-and-return” delay was chosen to give optimal excitation of the amino protons. The volume integral for each cross-peak was integrated and normalized for the sinusoidal excitation profile of the jump-and-return pulse sequence. The normalized volume for the cross-peak between C7H5 and C7H6 was used as a reference (2.45 Å) for calculating the interproton distances involving exchangeable protons using the isolated spin pair approach. Due to the inherent error in this approach, upper and lower bounds of (1 Å were assigned to all distance restraints. An additional 0.8 Å was added to the upper bounds for restraints involving methylene protons, and 1.0 Å was added to upper bounds for methyl protons. A total of 28 distance restraints derived from the NOESY spectrum recorded in H2O were included in the RMD simulation. In addition, a few restraints with loose bounds were derived by inspection of the spectra in D2O to give a grand total of 408 distance restraints incorporated into the RMD simulations (Table 4). Dihedral angle restraints for the H1′-C1′-C2′-H2′, H1′-C1′-C2′-H2′′, H2′-C2′-C3′-H3′, and H2′′-C2′C3′-H3′ deoxyribose angles were obtained by the angles returned for the most populated conformation by PSEUROT. Upper and lower bounds of (3° were assigned giving a flat well potential width of 6°. A total of 20 dihedral angle restraints were included in the RMD simulations. Twenty final structures each were generated for the TOTOEt complex starting from either an A-form or B-form DNA. All the structures converged to one family of conformations. The root-mean-square (rms) deviation of the coordinates of the 40 structures was 0.52 Å. The sum of violations was 11.4 Å for the A-form starting structure and 10.9 Å for the B-form starting structure. We observed three violations of >0.3 Å with none being >0.6 Å. These violations involved backbone H5′ and H5′′ protons of A5, perhaps an indication of unresolved


a

b

Figure 2. Stick plot of one of the calculated TOTOEt-DNA structures showing the helix axis. (a) View from the side of the helix. (b) View perpendicular to the view in penel a. Deoxyribose protons have been omitted for clarity.

dynamics of the phosphate backbone of the central part of the oligonucleotide. Different views of the structure are presented in Figures 2-4. The reliability of the structure calculations was carefully evaluated. RMD calculations in which the force constants of the distance restraints, k1, were lowered to 25 kcal/Å2 generated structures that converged to exactly the same family of structure as with the higher force constants, although the rmsd was slightly higher. The structure obtained is not strongly dependent on the exclusion of any of the restraints. In particular, calculations performed without including the Watson-Crick restraints yielded exactly the same structure, although the rmsd value was slightly higher. Helix Parameters. Helical parameters for the 40 final structures were thoroughly analyzed with the program CURVES 5.1 (Lavery and Sklenar, 1988, 1989). The CURVES algorithm allows the determination of local structural parameters and the overall helical axis for irregular nucleic acid structures. Plots of some of the global helical parameters for the TOTOEt complex are shown in Figure 5. The structural parameters for canonical A-DNA and B-DNA are included in Figure 5 for comparison. DISCUSSION

Binding Mode and Selectivity of TOTOEt. The 1D NMR spectrum of TOTOEt bound to d(CGCTAGCG)2 exhibits sharp lines (line widths ≈ 7 Hz) indicating that there is no chemical exchange between different binding sites. TOTOEt binds to the (5′-CTAG-3′)2 site by bisintercalating in the CT and AG base steps with an affinity more than 100 times higher than that to any other possible site in the oligomer. There seems to be some evidence that the binding of TOTOEt to dsDNA is even more selective and tighter than that to TOTO. The chemical shift values of the dsDNA protons at the binding site in the TOTOEt complex reflect intercalation of the aromatic chromophores. Compared to the free dsDNA, the T4 CH3(6) protons are shifted 0.8 ppm upfield. This is in agreement with the structure of the complex that shows that this methyl group is positioned exactly above the benzothiazole ring and thus exposed to a large ring current effect. Similar strong shifts are observed for A5 H1′, A5 H2′′, C3 H6, and T4 H6, consistent with the positions of these protons either above 1H


or below a ring system of the intercalating chromophore or near the intercalation site. Description of the Structure. The structure of the TOTOEt-dsDNA shows that TOTOEt bis-intercalates in the (5′-CTAG-3′)2 site with the benzothiazole ring system sandwiched between the pyrimidines and the quinolinium ring system between the purines. The N-ethyl group on the benzothiazole is placed in the major groove pointing toward the center of the oligonucleotide and the dyad symmetry axis of the complex to avoid a steric clash with the thymidine methyl group. The linker between the two chromophores is positioned in the minor groove, crossing from one side of the groove to the other. This is similar to what has been observed in other TOTO analogues (Spielmann et al., 1995b; Stærk et al., 1997). In the complex, the TOTOEt chromophores extend across the dsDNA helix. T4 and T12 unstack from A5 and A13 and move into the major groove to maximize van der Waals contacts with the benzothiazole ring of TOTOEt. The thymidine methyl groups are positioned right on top of the benzothiazole ring of TOTOEt. The observed imino proton cross-peaks, including the crosspeaks to the adenine amino protons and interstrand cross-peaks between adenine H2 and thymidine H1′, prove the existence of normal Watson-Crick base pairing in the intercalation site. So even though the thymidines are unstacked from the neighboring adenines, the base pairings of the thymidines with the complementary adenines do not appear to be appreciably disturbed. This is similar to what was observed in the TOTO complex (Spielmann et al., 1995b). Sugar Conformations. In B-form DNA, the deoxyribose ring adopts an S-type conformation described by a pseudorotation angle P of 180 ( 90°, while the deoxyribose residues of A-form DNA adopt an N-type conformation described by a pseudorotation angle P of 0 ( 90°. The results from the PSEUROT calculations resulted in S-type sugar conformations for all residues analyzed except for C3 in a purity above ∼80% with the pseudorotation angle PS between 120 and 175° corresponding to C1′-exo and C2′-endo conformations, respectively. For the C3 sugar part, no minimum could be found by the PSEUROT procedure, indicating that the dynamical processes governing the C3 sugar conformation are complex and cannot be described by the two-state model applied by PSEUROT. The C3 nucleotide is flanking one of the intercalated TOTOEt chromophores on the 5′ side. Similar complex dynamics were found for the nucleotides flanking the intercalation side of psoralen monoadducted and cross-linked to a DNA oligomer (Emsley et al., 1993). It should be noted, though, that the sugars of the two nucleotides trapped between the TOTOEt chromophores are found to be in relatively pure S-type conformations (Table 2). Helix Parameters. There are three major categories of helical parameters: axis-base pair, intra-base pair, and inter-base pair parameters (Dickerson et al., 1989). These parameters are discussed for the TOTOEt complex with respect to the calculated global helix axis below. However, the parameters reported for the terminal base pairs of the complex are not reliable because of dynamic processes and are included only for completeness. Spectroscopically, the complex was found to possess C2 symmetry. The small deviation of the structure and the calculated helical parameters from this symmetry reflects the level of uncertainty in the data. Global Helix Axis. The global helix axis in the dsDNA-TOTOEt complex is not straight but exhibits a pronounced kink at the intercalation site (Figure 2). The


a


b

Figure 3. Stereoview of a superposition of 10 randomly chosen structures of the 40 structures obtained by RMD calculations for the TOTOEt-DNA complex. Deoxyribose protons have been omitted for clarity.

Figure 4. Stacking of the TOTOEt chromophore in the CpT‚ GpA binding site viewed from the side. Only nucleobases and the TOTOEt chromophores are shown. It is evident that the benzothiazole N-ethyl group protrudes into the major groove. The following coloring scheme is used: TOTOEt chromophore, red; benzothiazole N-ethyl, yellow; adenine base, green; and thymine base, blue.

helix axis can be divided into different parts. The T4A5 segment is almost parallel to the terminal parts at both the 5′ and 3′ ends of the helix, while the intermediate parts, G2-T4 and A5-C7, describe a slip dislocation of these segments caused by the bis-intercalated TOTOEt. The effects of the slip dislocations of the helix axis are demonstrated by the helical parameters. Besides the pronounced kink, the helix axis of the TOTOEt complex shows a small bend (∼5°) into the minor groove. We believe that the kink of the global helix axis in the dsDNA-TOTOEt complex is not a mere artifact of the

structure determination. We support this conclusion with the fact that the artifact of distance restraints that are too short applied in the RMD protocol normally produces bends into the major groove due to the larger density of restraints in the major groove of dsDNA (Spielmann et al., 1995a). Furthermore, the distribution of restraints applied in the structure determination of the TOTOEt complex is fairly isotropic around the intercalation site, diminishing the effects of foul distance restraints. The kink of the helix axis occurs around the TOTOEt intercalation site, and we believe the benzothiazole N-ethyl group is responsible for this as the dsDNATOTO complex shows no kinks or bends of the helix axis. It has recently been demonstrated that the placement of bulky groups in the minor groove of dsDNA can induce pronounced bending into the major groove (Chen et al., 1996). The benzothiazole ring system does not stack exactly coplanar with T4 but is twisted slightly relative to the nucleobase and thus inducing the kink in the helix axis. Axis-Base Pair Parameters. X-axis displacement, Yaxis displacement, inclination, and tip are the parameters minimized to obtain the global helix axis (Lavery and Sklenar, 1988). These parameters thus describe how the base pairs are positioned in the helix relative to the global helix axis. The X-axis displacement and Y-axis displacement are both close to the values for B-DNA, while inclination shows significant deviations with an evident C2 symmetry. The value for tip shows large and correlated changes from the B-DNA values, changing from negative to positive going from C3 to G6; this is a direct consequence of the geometry of the kinking helix axis. Intra-Base Pair Parameters. The values obtained for shear, stretch, stagger, and opening are partly determined by the constraints introduced to mimic WatsonCrick base pairing. However, in the central part of the oligonucleotide, the numerous dsDNA-TOTOEt NOEs



Figure 5. Helical parameters for the TOTOEt complex calculated using CURVES and compared with those of canonical A-DNA (‚‚‚) and B-DNA (- - -).

lock the conformation of the structure. The TOTOEt ligand extends across the duplex and hence makes NOE contacts to both strands. This contributes significantly to the determination of the above parameters.

The shear changes from negative to positive across the intercalation site, compensating for the intercalation of the TOTOEt chromophores. The shear introduced possibly stems from the A5 maximizing the overlap with one


ring of the quinolinium ring system and the T4 moving into the major groove to maximize van der Waals forces with the benzothiazole ring system. There is only a slight net shear introduced in the helix. This was also observed for the dsDNA-TOTO structure (Spielmann et al., 1995b). The stretch is essentially that of B-DNA. Base opening is almost symmetrically disposed around the T4-A5 base step. An opening angle larger than that of standard DNA narrows the minor groove. It is seen that the T4 moves into the major groove and the minor groove subsequently narrows, probably also due to van der Waals interactions between the linker N-methyl groups and electrostatic attractions between the anionic DNA phosphates and the linker nitrogens. The opening observed for the central base pairs is not as large as that found in the dsDNA-TOTO complex. This is a consequence of the near coplanarity of the two ring systems of the TOTOEt chromophores. For the TOTO complex, it was found that the chromophores were twisted about the quinoline-C8-thiazole bond by an angle of 22°. In the dsDNA-TOTOEt complex, this angle is 6°. A GAUSSIAN94 calculation performed on the free TOTOEt shows an angle of 22° between the two planes of the chromophores. The decrease of this angle from the free TOTOEt to the bound is due to clamping of TOTOEt by the nucleobases in the complex. The slight twisting of the TOTOEt chromophores is mirrored in the parameters for buckle and propeller twist. It is reported that intercalation can induce buckling in dsDNA as the base pairs adjacent to the intercalation site buckle to maximize van der Waals contacts with the chromophore(s) of the intercalating agent (Williams and Gao, 1992). TOTOEt introduces almost no buckle in the dsDNA, indicating that the nucleobases of the complex can maximize van der Waals contacts and stacking with the TOTOEt chromophores without the propensity to introduce buckling. The propeller twists of the two central base pairs are essentially that of B-DNA. A large propeller twist is introduced into the C3‚G14 and G6‚C11 base pairs. This is because the G6‚G14 remains coplanar with the quinolinium ring system, while C3‚C11 stacks with G2‚G10. Inter-Base Pair Parameters. The roll, slide, and shift in regular A-DNA and B-DNA conformations are essentially zero. These parameters show large and correlated changes from the equilibrium values in the TOTOEt complex. There are shifts for the two intercalation sites with opposite signs that cancel each other in terms of the overall helix. The slide exhibits correlated and compensatory changes for the C3‚T4 and T4‚A5 base pair steps to accommodate the dye intercalation. The shift and slide values are exact mirrors of the lateral displacements of the global helix axis. The central base pair step T4‚A5 has a positive roll. Positive roll opens the angle between base pairs toward the minor groove, resulting in a bend into the major groove. A negative roll results in a bend into the minor groove. The central base pair T4‚A5 is clamped between the two thiazole orange chromophores to maximize van der Waals contacts. The bases roll to open the minor groove. The base pair steps flanking the intercalation sites show almost zero rolls, while the G2‚C3 and G6‚C7 base pair steps one step away from the intercalation site exhibit significant negative roll. This is to compensate for the kinking of the helix axis at the C3‚T4 and A5‚G6 base pair steps. The helix is unwound by 80° and has an overall helical repeat of 12 base pairs in the complex, consistent with the bis-intercalation of TOTOEt. It is noteworthy that


the unwinding is largely confined to the two base pair steps defining the intercalation site. CONCLUSION

The results obtained in this work show that TOTOEt binds selectively to dsDNA with (5′-CTAG-3′)2 as the preferred binding site. Compared to TOTO, this implies that modification of this drug on the benzothiazole N atom system is possible while still maintaining or even enhancing the selectivity and binding affinity. Our study showed that an extremely well defined complex between TOTOEt and (5′-CGCTAGCG-3′)2 was obtained. The complex seems to be even more well defined than that in the case of TOTO, indicating that TOTOEt has a higher affinity and selectivity toward dsDNA. This is possibly caused by the benzothiazole N-ethyl group clamping the drug to the dsDNA. The N-ethyl group on the benzothiazole of TOTOEt is placed in the major groove pointing toward the center of the oligonucleotide and the dyad symmetry axis of the complex. This is exactly the orientation that is required for a TOTO analogue with a linker connecting the two chromophores in the major groove. Consequently, it seems rather straightforward to design such an analogue, the only remaining problem being the length of the linker. Direct measurement of the structure indicates the linker in the major groove should be slightly shorter than the linker in the minor groove. After the proper linker in the major groove has been found, it seems obvious from the structure that the subsequent design of a TOTO analogue with a linker in both the major groove and the minor groove is straightforward. This would probably yield a selective dsDNA binding drug with an extremely high binding affinity and vanishing kinetic abilities. ACKNOWLEDGMENT

We are grateful to Dr. E. B. Pedersen (Department of Chemistry, Odense University, Denmark) for providing TOTOEt. Professor H. Peter Spielmann is acknowledged for supporting this work with spectrometer time on a Varian Unity+ 500 NMR spectrometer and many helpful discussions. LITERATURE CITED Bishop, J. M. (1987) The Molecular Genetics of Cancer. Nature 235, 305-311. Boelens, R., Scheek, R. M., Dijkstra, K., and Kaptein, R. (1985) Sequential Assigment of Imino- and Amino-Proton Resonances in 1H NMR Spectra of Oligonucleotides by TwoDimensional NMR Spectroscopy. Application to lac Operator Fragment. J. Magn. Reson. 62, 378-386. Borgias, B. A., and James, T. L. (1990) MARDIGRAS-A procedure for matrix analysis af relaxation for discerning geometry of an aqueous structure. J. Magn. Reson. 87, 475-487. Borgias, B. A., Gochin, M., Kerwood, D., and James, T. L. (1990) Relaxation Matrix analysis of 2D NMR data. Prog. NMR Spectrosc. 22, 83-100. Chen, H., Liu, X., and Patel, D. J. (1996) DNA Bending and Unwinding Associated with Actinomycin D Antibiotics Bound to Partially Overlapping Sites on DNA. J. Mol. Biol. 258, 457-479. de Leeuw, F. A. A. M., and Altona, C. (1983) Computer-Assisted Pseudorotation Analysis of Five-Membered Rings by Means of Proton Spin-Spin Coupling Constants: Program PSEUROT. J. Compt. Chem. 4, 428-437. Dickerson, R. E., Bansal, M., Calladine, C. R., Diekmann, S., Hunter, W. N., Kennard, O., von Kitzing, E., Nelson, H. C. M., Lavery, R., Olson, W. K., Saenger, W., Shakked, Z., Soumpasis, D. M., Tung, C.-S., Sklenar, H., Wang, A. H. J.,

340 Bioconjugate Chem., Vol. 9, No. 3, 1998 and Zhurkin, V. B. (1989) Definitions and nomenclature of nucleic acid structure parameters. EMBO J. 8, 1-4. Emsley, L., Dwyer, T. J., Spielmann, H. P., and Wemmer, D. E. (1993) Determination of DNA Conformational Features from Selective Two-Dimensional NMR Experiments. J. Am. Chem. Soc. 115, 7765-7771. Faridi, J., Nielsen, K. E., Stein, P. C., and Jacobsen, J. P. (1997) Bis-Intercalation of a Homodimeric Thiazole Orange Dye in DNA: Dynamic Exchange between Two Sites. J. Biomol. Struct. Dyn. 15, 321-331. Feigon, J., Leupin, W., Denny, W. A., and Kearns, D. R. (1983) Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2. Biochemistry 22, 5943-5951. Gochin, M., Zon, G., and James, T. L. (1990) Two-Dimensional COSY and Two-Dimensional NOE Spectroscopy of d(AC)4‚ d(AC)4: Extraction of Structural Constraints. Biochemistry 29, 11161-11171. Gue´ron, M., and Plateau, P. (1982) Exchangeable Proton NMR without Base-Line Distortion, Using New Strong-Pulse Sequences. J. Am. Chem. Soc. 104, 7310-7311. Hansen, L. F., Jensen, L. K., and Jacobsen, J. P. (1996) Bisintercalation of a homodimeric thiazole orange dye in DNA in symmetrical pyrimidine-pyrimidine-purine-purine oligonucleotides. Nucleic Acids Res. 24, 859-867. Hare, D. R., Wemmer, D. E., Chou, S.-H., Drobny, G., and Reid, B. R. (1983) Assignment of the nonexchangeable proton resonances of d(C-G-C-G-A-A-T-T-C-G-C-G) using two-dimensional nuclear magnetic resonance methods. J. Mol. Biol. 171, 319-336. Jacobsen, J. P., Pedersen, J. B., Hansen, L. F., and Wemmer, D. E. (1995) Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides. Nucleic Acids Res. 23, 753-760. Lavery, R., and Sklenar, H. (1988) The definition of generalized helicoidal parameters and for axis curvature of irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63-91. Lavery, R., and Sklenar, H. (1989) Defining the structure of irregular nucleic acids: Conventions and principles. J. Biomol. Struct. Dyn. 7, 655-667.

Petersen and Jacobsen Liu, H., Spielmann, H. P., Ulyanov, N. A., Wemmer, D. E., and James, T. L. (1995) Interproton distances bounds from 2DNOE intensities: Effect of experimental noise and peak integrations errors. J. Biol. NMR 6, 390-402. Macaya, R. F., Schultze, P., and Feigon, J. (1992) Sugar Conformation in Intramolecular DNA Triplexes Determined by Coupling Constants Obtained by Automated Simulation of P.COSY Cross Peaks. J. Am. Chem. Soc. 114, 781-783. Scheek, R. M., Russo, N., Boelens, R., and Kaptein, R. (1983) Sequential Resonance Assigments in DNA 1H NMR Spectra by Two-Dimensional NOE Spectroscopy. J. Am. Chem. Soc. 105, 2914-2916. Scheek, R. M., Boelens, R., Russo, N., Van Boom, J. H., and Kaptein, R. (1984) Sequential Resonance Assigments in 1H NMR Spectra of Oligonucleotides by Two-Dimensional NMR Spectroscopy. Biochemistry 23, 1371-1376. Schmitz, U., and James, T. L. (1995) How to Generate Accurate Solution Structures of Double-Helical Nucleic Acid Fragments Using Nuclear Magnetic Resonance and Restrained Molecular Dynamics. Methods Enzymol. 261, 3-44. Spielmann, H. P., Dwyer, T. J., Hearst, J. S., and Wemmer, D. E. (1995a) Solution Structure of Psoralen Monoadducted and Cross Linked DNA Oligomers by NMR Spectroscopy and Restrained Molecular Dynamics. Biochemistry 34, 1293712953. Spielmann, H. P., Wemmer, D. E., and Jacobsen, J. P. (1995b) Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Determined by NMR Spectroscopy. Biochemistry 34, 8542-8553. Stærk, D., Hamed, A. A., Pedersen, E. B., and Jacobsen, J. P. (1997) Bis-Intercalation of Homodimeric Thiazole Orange Dyes in DNA: The Effect of Modifying the Linker. Bioconjugate Chem. 8, 869-877. Williams, L. D., and Gao, Q. (1992) DNA-Ditercalinium Interactions: Implications for Recognition of Damaged DNA. Biochemistry 31, 4315-4324. Wu¨thrich, K. (1986) in NMR of Proteins and Nucleic Acids, John Wiley & Sons, New York.

BC970133I

Solution Structure of a DNA Complex with the Fluorescent Bis

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