Structure of a DNA Duplex Containing a Single 2 '-O-Methyl-Î²-d-araT

Bioconjugate Chem. 1996, 7, 680−688

680

Structure of a DNA Duplex Containing a Single 2′-O-Methyl-β-D-araT: Combined Use of NMR, Restrained Molecular Dynamics, and Full Relaxation Matrix Refinement Charlotte H. Gotfredsen,† H. Peter Spielmann,‡ Jesper Wengel,† and Jens Peter Jacobsen*,† Department of Chemistry, Odense University, DK-5230 Odense M, Denmark, and Department of Biochemistry, University of Kentucky, Lexington, Kentucky 40536. Received June 24, 1996X

Two-dimensional 1H NMR spectroscopy was used to determine the solution structure of the doublestranded DNA oligonucleotide d(5′-CGCATATAGCC-3′): d(5′-GGCTAXATGCG-3′), where X is 1-(2O-methyl-β-D-arabinofuranosyl)thymine. The structure determination was based on a total relaxation matrix analysis of NOESY cross-peak intensities using the MARDIGRAS program. The improved RANDMARDI procedure was used during the calculations to include the experimental “noise” in the NOESY spectra. The NOE-derived distance restraints were applied in restrained molecular dynamics calculations. Twenty final structures each were generated for the modified DNA duplex from both A-form and B-form DNA starting structures. The root-mean-square deviation of the coordinates for the 40 structures was 0.82 Å. The duplex adopts a normal B-DNA-type helix, and the spectra as well as the structure show that the modified nucleotide X adopts a C2′-endo (S) sugar conformation. There are no significant changes in the helix originating from the modified nucleotide. The CH3O group on X is directed toward the major groove, and there seems to be free space for further modifications at this position.

INTRODUCTION

A potential gene inhibitor must possess an enhanced stability toward cellular nucleases, the ability to penetrate the cell membrane, and an efficient hybridization to the target RNA/DNA1 (He´le`ne and Toulme´, 1990; Uhlmann and Peyman, 1990). Several chemically modified DNA and RNA oligonucleotides have been synthesized, and a few of them fulfill some of these requirements (Inoue et al., 1987; Sproat et al., 1989; Egholm et al., 1993; J¢rgensen et al., 1994; Lesnik et al., 1993). Among these are 2′-O-methylated ribonucleotides (2′OCH3-RNA) (Figure 1), which are resistant toward DNAand RNA-specific nucleases and have improved hybridization properties. However, they are degradable by a dual RNA/DNA active enzyme such as snake venom phosphodiesterase (SV PDE) (Inoue et al., 1987; Sproat et al., 1989; Lesnik et al., 1993). Structural studies on oligodeoxynucleotides containing the 2′-O-methylated ribonucleotides show that introduction of only one modified nucleotide causes a local change in the DNA conformation (Lubini et al., 1994). A CH3O group at the 2′ribo position on a single residue or an all-modified strand causes changes from a B-DNA to a A-DNA-type conformation (Lubini et al., 1994; Blommers et al., 1994) and forces it into a C3′-endo sugar pucker conformation * Author to whom correspondence should be addressed. † Odense University. ‡ University of Kentucky. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: AMBER, assisted model building with energy refinement; DNA, deoxyribonucleic acid; dsDNA, doublestranded DNA; EDTA, ethylenediaminetetraacetic acid; 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; SV PDE, snake venom phosphodiesterase; TAXA, d(5′-CGCATATAGCC3′):d(5′-GGCTAXATGCG-3′); TOCSY, total correlation spectroscopy; TPPI, time proportional phase incrementation.

S1043-1802(96)00061-4 CCC: $12.00

Figure 1. Structure of 1-(2-O-methyl-β-D-arabinofuranosyl)thymine and 2′-OCH3-RNA together with their predominant sugar conformations. B denotes a nucleobase.

(Guschlbauer and Jankowski, 1980). The structure of an araC-modified double-stranded oligonucleotide has been reported in the literature with significant structural changes at the modification site compared to the unmodified oligonucleotide (Schweitzer et al., 1994; Gao et al., 1991). Recently, we have described the design and synthesis of an oligonucleotide containing the thymidine analog 1-(2-O-methyl-β-D-arabinofuranosyl)thymine (2′-OCH3araT, X) (Figure 1) (Gotfredsen et al., 1994, 1996). The aim of this work is fourfold: (i) to minimize the structural distortions caused by the modification relative to the B-DNA structure of an unmodified duplex, (ii) to maintain normal Watson-Crick sequence selectivity of the modified oligonucleotide, (iii) to create an unhindered point of attachment for functional groups oriented into the major groove of regular B-form DNA, and (iv) to enhance the nuclease resistance of the oligonucleotide. In this work we have used NMR spectroscopy to evaluate the success of the design of the modified oligonucleotide containing a 2′-OCH3-araT nucleotide. We have determined the solution structure of the undecanucleotide duplex d(5′-CGCATATAGCC-3′):d(5′-GGCTAX© 1996 American Chemical Society

1H

Bioconjugate Chem., Vol. 7, No. 6, 1996 681

NMR Structure Determination of a Modified Oligonucleotide

ATGCG-3′) (TAXA) using 1H NMR. Data from NOESY spectra were used to obtain interproton distance bounds using the RANDMARDI procedure (Liu et al., 1995), an improved version of the MARDIGRAS complete relaxation matrix approach (Borgias et al., 1990; Borgias and James, 1990). These distance bounds were used as restraints in molecular dynamics (RMD) calculations. Since many NOE contacts were observed between the modified nucleotide and the surrounding DNA, the resulting structures have fairly high resolution and allowed the determination of some local conformational features in the DNA. EXPERIMENTAL PROCEDURES

Sample Preparation. The synthesis of oligonucleotides containing the thymidine analog 1-(2-O-methylβ-D-arabinofuranosyl)thymine (2′-OCH3-araT, X) has previously been described (Gotfredsen et al., 1994, 1996). A standard phosphoramidite method on a Pharmacia Gene Assembler Special DNA synthesizer was used. The crude product was purified by ethanol precipitation. The numbering scheme for the double-stranded oligonucleotide is

The duplex was formed by mixing (1:1) and annealing the complementary single-stranded oligomers at 80 °C and cooling to room temperature over a 2 h period. The samples of the modified and unmodified oligonucleotides were prepared by dissolving the lyophilized complex in 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0) and 0.05 mM sodium EDTA. For experiments carried out in D2O the samples were lyophilized twice from D2O on a SpeedVac evaporator and redissolved in 99.96% D2O (Cambridge Isotope Laboratories). A 90% H2O/10% D2O mixture was used to obtain NMR spectra of exchangeable protons. The dsDNA concentrations of the modified and unmodified oligonucleotide were 1.7 and 2.0 mM, respectively. UV and NMR studies were performed to compare the relative stabilities of the unmodified and the modified oligonucleotides as reported elsewhere (Gotfredsen et al., 1994, 1996). NMR Experiments. NOESY and TOCSY NMR spectra were obtained on a Varian Unity 500 spectrometer. The NOESY buildup experiments in D2O of TAXA were acquired with mixing times of 25, 50, 100, 150, and 200 ms. The States phase cycle scheme was used to obtain 512 t1 experiments; 64 scans were acquired for each t1 value using 1024 complex points in t2, a spectral width of 5000 Hz, and a recycle delay of 2 s. These NOESY spectra were obtained repetitively without removing the sample from the magnet. The TOCSY spectra were obtained in D2O with mixing times of 30, 90, and 140 ms in the TPPI mode using 1024 complex points in t2 and 512 t1 experiments each acquired with 80 scans. The spectral width was 5000 Hz. NOESY spectra in H2O with mixing times 100 and 200 ms were obtained with a NOESY pulse sequence in which the last 90° pulse was replaced by a jump and return sequence (Gue´ron et al., 1991) to suppress the solvent signal. The spectra were acquired with a spectral width of 10 000 Hz in 2048 complex points using 512 t1 experiments with 64 scans each. The data were processed using FELIX (version 2.1, Biosym Technologies, San Diego). Structure Calculation. The upper and lower halves of the diagonal of each of the five assigned NOESY spectra were integrated separately with FELIX. The

integrated volumes of symmetry-related cross peaks above and below the diagonal were treated as independent measurements to more accurately reflect the scatter in the data. This gave a total of 10 peak intensity sets, 2 for each of the five mixing times. The spin-lattice relaxation times were measured for representative protons by the inversion recovery method. The measured intensities of the NOESY cross peaks were scaled for the effects of incomplete relaxation. The RANDMARDI procedure (Liu et al., 1995) of the complete relaxation matrix analysis method, MARDIGRAS (Borgias and James, 1990; Borgias et al., 1990; Schmitz et al., 1992; Schmitz and James, 1996), was used to calculate interproton distance RMD bounds from the resulting integrated peak intensities. The RANDMARDI procedure was used to include the effects of experimental noise in the relaxation matrix calculations. RANDMARDI adds a random number from within a specified range to each input intensity used in a MARDIGRAS calculation. The noise level used for the TAXA sequence was 0.65% of the largest integrated cross peak or ∼5 times the intensity of the smallest peak. 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 10 data sets. The bounds files from all five mixing times are then combined into a single bounds file from which the RMD restraint file is generated. Upper and lower bounds were average interproton distance ( 1 standard deviation calculated from all of the MARDIGRAS runs. The RANDMARDI procedure accurately accounts for the effects of random experimental noise and integration errors. A complete description of the RANDMARDI theory and procedure is given elsewhere (Liu et al., 1995). The distance restraints obtained from the MARDIGRAS calculation were incorporated into a restrained molecular dynamics (RMD) procedure. The RMD and energy minimization calculations were performed using DISCOVER (version 2.95) with the AMBER force field potentials, and the models were displayed using INSIGHTII (version 2.3.0) (Biosym Technologies). A starting model of either A-form or B-form DNA was built using standard Arnott parameters (Biosym Technologies), and the H2′ proton of the X17 residue was replaced with a methoxy group using the BUILDER module of INSIGHTII. The NOE-derived distance restraints were then applied, and the model was energy minimized to a maximum derivative of 0.1 Å. This was followed by 19 ps of RMD with the following temperature profile: 450 K for 4 ps cooled 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 term used to enforce the distance restraints was

{

) k1(r - r2)2

Econstr ) 0 ) k2(r - r3)2

when r2 > r when r3 g r g r2 when r3 > r

where r2 and r3 are the upper and lower distance bounds determined from the cross-peak volumes, and k1 and k2 are the force constants that can be independently adjusted for each restraint. An upper and lower bound force constant of 50 kcal/mol Å2 was assigned for all NOEderived distance restraints; a set of calculations with a

682 Bioconjugate Chem., Vol. 7, No. 6, 1996

Gotfredsen et al.

Table 1. Chemical Shifts (ppm) for the TAXA Sequencea TAXA

H(1′)

H(2′)

H(2′′)

H(3′)

H(4′)

H(5′)

H(5′′)

H(6)/H(8)

H(5)/H(2)/CH3

C1 G2 C3 A4 T5 A6 T7 A8 G9 C10 C11 G12 G13 C14 T15 A16 X17 A18 T19 G20 C21 G22

5.78 5.91 5.63 6.27 5.80 6.13 5.64 6.01 5.74 5.98 6.24 5.71 5.97 5.98 5.76 6.32 5.63 6.16 5.76 5.82 5.78 6.15

1.95 2.65 2.06 2.70 2.19 2.48 1.99 2.67 2.50 2.12 2.24 2.52 2.52 2.07 2.19 2.65 3.54e 2.59 1.99 2.58 1.90 2.60

2.39 2.74 2.41 2.96 2.59 2.84 2.40 2.85 2.60 2.44 2.24 2.70 2.73 2.53 2.59 3.06 4.15 2.92 2.40 2.65 2.33 2.35

4.69 4.97 4.85 5.01 4.88 4.93 4.87 5.02 4.93 4.75 4.54 4.83 4.99 4.75 4.91 5.05 4.69 4.99 4.70 4.96 4.81 4.67

4.16 4.35 4.19 4.44 4.24 4.38 4.16d 4.37 4.36 4.16 4.03 4.18 4.41 4.26 4.19 4.46

3.71 3.74b

3.71 3.98b 4.22 4.16b 4.29

7.63 7.95 7.37 8.33 7.19 8.08 7.14 8.12 7.63 7.40 7.68 7.85 7.85 7.42 7.41 8.37 7.14 7.93 7.06 7.84 7.34 7.95

5.88

4.12b

3.67

4.14b

4.35 4.16d 4.35 4.16 4.16

4.22 4.06 4.20 4.36 4.12c 3.67 4.09 4.10 4.19b 4.39 4.22 4.25 4.07

H4/H6

H4/H6

5.42 7.62 1.52 6.85 1.22 7.21

8.31

6.43

5.29 5.76

8.14

6.41

5.29 1.63 7.22 1.43 7.07 1.25

8.14

6.50

5.39

8.38

H1/H3 13.01 13.23 13.28 12.79

13.00 13.57 13.00 13.46 12.59 6.52

a The values are given at 25 °C relative to DSS. Hydrogen-bonded amide protons are underlined. b We are unable to assign H5′ and H5′′ specifically; they may be interchanged. c This could be a H5′ proton instead of a H5′′ proton. d We are unable to decide whether it is T7H4′, T19H4′, or both overlapping. e This is the value of the 2′-OCH3.

force constant of 25 kcal/mol Å2 was also performed but without any changes in the output structure. An additional 28 distance restraints were included to enforce 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 five A-T base pairs with lower and upper bounds of 1.74 and 2.10 Å, respectively. Helix parameters were calculated with the program CURVES 3.1 (Lavery and Sklenar, 1988, 1989). RESULTS

Spectral Analysis. The line widths of resonances observed in the 1H NMR spectra of TAXA are similar to the ones found for the unmodified double-stranded oligonucleotide. The X17 2′-OCH3 group resonates at 3.54 ppm in a region of the spectrum normally devoid of DNA signals (Figure 2). The assignment of the nonexchangeable protons in both the unmodified undecamer, d(5′-CGCATATAGCC3′):d(5′-GGCTATATGCG-3′) and the 2′-OCH3-araT-modified TAXA undecamer was performed using standard methods (Hare et al., 1983; Wu¨thrich, 1986; Scheek et al., 1983). Aromatic (H6, H5, H8, and H2) and sugar protons (H1′, H2′, H2′′, H3′, most H4′, and some of the H5′, H5′′) were assigned. The assigned resonances of the modified oligonucleotide are given in Table 1. The assignments of the exchangeable protons were obtained from the NOESY spectrum in H2O (Wu¨thrich, 1986; Boelens et al., 1985). The chemical shift values and the NOE connectivity pattern of the imino protons were observed to be in accordance with normal Watson-Crick base pairing. Except for the 2′-OCH3-araT nucleotide there are no significant differences in the chemical shift values between the unmodified undecamer duplex and the modified TAXA duplex. The NOESY spectrum exhibits a few characteristic features originating from the 2′-OCH3-araT. Figure 2 shows the sequential connectivities from aromatic H6/ H8 protons to both intra- and inter-residue H2′/H2′′. The connectivites from the modified X17 OCH3/H2′′ to A18 H8 are shifted downfield compared to the normal deoxyribose H2′/H2′′. The presence of weak NOESY cross peaks from the H6/H8 base protons to H1′ protons

Figure 2. H2′/H2′′ to aromatic part of the 200 ms NOESY spectrum of TAXA. A solid line indicates the sequential connectivity pathway from X17 H2′′ and OCH3 to X17H6 and A18H8 and from X17H6-X17CH3 and X17CH3-A16H8.

indicates that the nucleobases are in an anti conformation. Furthermore, the NOESY spectra show very weak cross peaks from H3′ protons to base protons (data not shown), and only two strong cross peaks appear in the base proton to H2′/H2′′ region [(n) H6/H8 to (n)H2′ and (n) H6/H8 to (n - 1) H2′′] at short mixing times. This

1H



Figure 3. Graphical representation of the distribution of restraints for the structure calculations.

Figure 4. Structure of the modified DNA looking into the minor groove (left) and the major groove (right). X17 is shown in yellow, the nucleobases are in blue, and the sugar phosphate backbone is in red.

shows that all of the sugars adopt a C2′-endo (S)-type conformation. Structure Calculations. More than 445 NOE cross peaks were observed in the NOESY spectrum obtained with a mixing time of 200 ms. 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 254 NOESY cross peaks for each mixing time were used in the total relaxation matrix analysis. Integrations of the cross peaks in the five NOESY spectra used were done separately for each side of the diagonal. This yielded a total of 10 sets of measured NOE intensities that were converted to distance restraints using the RANDMARDI procedure. Some of the cross peaks used in the structure calculations resulted predominantly from spin diffusion and were consequently not observed in NOESY spectra with shorter mixing times. This is taken into account during the statistical analysis of the results generated by the RANDMARDI procedure (Liu et al., 1995). Cross-peak integrals that corresponded to fixed distances in the DNA were not converted into distance restraints for the use in the RMD simulations. The calculations returned 241 interproton distances that are not covalently fixed in the modified DNA. The MARDIGRAS calculations have been performed by using a value of the correlation time of τc ) 2 ns. This

value was justified from approximate values of τc calculated from measured spin-lattice relaxation times, T1, and spin-spin relaxation times, T2 (Woessner, 1962). The values of T1 were obtained from a nonselective inversion recovery experiment, whereas the values of T2 were calculated from the line widths of corresponding wellseparated lines of representative protons, such as methyl group protons, sugar protons, and aromatic protons including adenine H(2)s. Values for τc were found to be between 1.9 and 2.4 ns. Although this is only an estimated range of values, it justifies the use of τc ) 2 ns in the MARDIGRAS calculation. Furthermore, it turns out that the results of the calculations are rather insensitive to the exact values of τc used. This is similar to what was found earlier (Mujeeb et al., 1993). The distance bounds used in RMD simulations were determined by combining the results from all of the individual MARDIGRAS calculations performed during the RANDMARDI procedure into one set. They were calculated individually for each proton pair corresponding to a NOESY cross peak included in the RANDMARDI calculations. Each individual restraint was generated from the MARDIGRAS calculations using the calculated average distance ( 1 standard deviation. Figure 3 gives a summary of the distance restraints used in the RMD calculations. The range of differences between the upper and lower bounds for the 241 NOE-derived restraint bounds used (Figure 3) was between 0.08 and 3.59 Å with an average flat well potential width of 1.02 Å. This range between the upper and lower bounds represents the precision of the restraint calculated from the NOESY data. Weak NOESY cross peaks primarily from spin diffusion had the largest distance between the upper and lower distance bounds. Evaluation of the spectra obtained in H2O shows normal Watson-Crick hydrogen bonding, justifying the inclusion of 28 hydrogen bond distance restraints. An additional 10 restraints with loose bounds were derived by inspection of the spectra for a total of 279 interproton restraints and used in the RMD calculations. The distribution of restraints (Figure 3) is anisotropic, with the central part of the duplex being the most well defined. Twenty final structures were generated for the modified duplex starting either from a B-form or an A-form DNA conformation. All of the structures converged to one family of conformations. The root-mean-square (rms) deviation of the coordinates of the 40 structures was 0.82


Gotfredsen et al.

Figure 5. Stereoview of the stick plot of the structure of TAXA. Deoxyribose protons have been omitted for clarity except for X17. (Top) One of the calculated structures with an indication of the direction of the helix axis obtained from CURVES. (Bottom) A superposition of 10 of the 40 structures obtained by RMD calculations. The 10 structures are randomly picked from structures calculated from both A- and B-DNA starting structures.

Sklenar, 1988, 1989), and selected parameters are shown in Figure 8. The structural parameters for canonical A-DNA and B-DNA are included in Figure 8 for comparison (Arnott and Hukins, 1972, 1973). DISCUSSION

Figure 6. View of the OCH3 (yellow) in the major groove and the close proximity of it to A18H8 (yellow). The nucleobases are shown in blue and the sugar phosphate backbone is in red.

Å. The sum of residual violations of the distance restraints for the structures shown was 7.6 Å for the B-form starting structure and 7.3 Å for the A-form starting structure. We had five violations >0.2 Å, with no violations >0.29 Å. Different views of the structure are given in Figures 4-6. Helical parameters for the 40 final structures were calculated with the program CURVES 3.1 (Lavery and

Description of the Structure. Qualitative analysis of the NOESY spectra indicates that TAXA adopts a B-form conformation. This is confirmed by the structure calculation. All bases are in an anti conformation, and they form normal Watson-Crick base pairs. The structure shows that the CH3O group is in the major groove. The spectral analysis and the structure calculation give no indications of different families of O-methyl rotamers about the C2′-O2′ methoxy bond, and there are no mutually inconsistent NOEs observed from the X17 2′OCH3 group to the surrounding DNA. There is no sign of exchange peaks to suggest flipping between different positions. The calculations give only one family of

1H



Figure 7. Superimposition of the middle ATATA part of TAXA with the ATATA part of d(CGCATATATGCG)2 taken from the Brookhaven databank. In the figure the deoxyribose protons are removed for clarity.

structures. The X17 2′-OCH3 is in van der Waals contact with A18 H8 (Figure 6) (2.95-3.28 Å). Experimentally derived NMR restraints are distributed anisotropically and short range in nature. This largely establishes local structures with some helical parameters better defined by the experimental data than others. We have found a number of NMR-derived restraints in the center of the helix originating from AH2 protons to H1′ protons. These NOE-derived distance restraints improved the ability to define the conformation for the central five bae pairs of the oligonucleotide (Weisz et al., 1994). Helix Parameters. There are three major categories of helical parameters: axis-base pair, intrabase pair, and interbase pair parameters (Dickerson et al., 1989). Some of these parameters for TAXA are shown in Figure 8. Error bars are not included since we found only very small variations in the calculated values. The global helix axis is bent toward the major groove (Figure 5) with an overall helix axis curvature of 12°. Some of the features of the calculated helix parameters with respect to the calculated global helix axis are discussed below. Values of the pseudorotational angles obtained from CURVES are within the range of 106-178° with the values for X17 at 145°. This is in agreement with the finding that TAXA adopts a B-like DNA conformation. Axis-Base Pair Parameters. The structure around the X-modification in TAXA has inclination values closer to those for B-DNA than for A-DNA. The variation of the inclination follows the width of the minor groove, being tightest around the modification site as compared to the most negative value for the inclination (Yuan et al., 1992). For TAXA the tip angle starts negative and changes to positive, but this is within the interval of (5° common for canonical A-DNA and B-DNA (Mujeeb et al., 1993). Intrabase Pair Parameters. The calculated values for shear, stretch, stagger, and opening are highly influenced by the restraints used to enforce base pair hydrogen bonding and are therefore not included in Figure 8. The buckle changes smoothly along the helix to a negative value of -16.5° at the modification site from positive values at each end of the helix axis. The large negative value at X shows that the center of this base pair is bent away from the center of the helix (Mujeeb et al., 1993). The propeller twist for all base pairs is negative. This is typical for structures determined in solution by NMR of other oligonucleotides (Baleja et al., 1990; Weisz et al., 1994; Mujeeb et al., 1993; Schmitz et al., 1992). Base pair opening values for TAXA are larger than those for canonical A- and B-form DNAs. This corresponds to a local expansion of the minor groove in the (5′-A4pT5-3′): (5′-A18pT19-3′) and (5′-T7pA8-3′):(5′-T15pA16-3′) regions. The minor groove is locally tightened in the region between. The width of the minor groove was measured

as the shortest interstrand phosphorus-phosphorus distance (i, i + 14) across the groove minus the combined van der Waals radii of the phosphorus atoms (5.8 Å). The tighter minor groove in the (5′-A6pT7pA8-3′):(5′-T15pA16pX17-3′) region (5.3-5.9 Å) is reflected in an observable NOE cross peak from A6 H2 and the A18 H1′ (Kintanar et al., 1987; Weisz et al., 1994). Interbase Pair Parameters. Values for the rise in the TAXA sequence are midway between those of canonical A-form and B-form. This is also reflected in the overall length of the helix, which is 32.8 Å. The roll parameter exhibits an alternating pattern along the duplex but with a significant positive roll value at the (5′-C3pA4-3′):(5′T19pG20-3′) site and at the (5′-T7pA8-3′):(5′-T15pA163′) site. Positive rolls open the angle between the base pairs toward the minor groove. As a result, a wider minor groove and bending toward the major groove occurs. In recent in vitro and NMR studies (5′-CpA-3′): (5′-TpG-3′) steps have been correlated with DNA bending (Beutel and Gold, 1992; Weisz et al., 1994; Mujeeb et al., 1993). Due to local bending from these positive roll values, the global helical curvature is affected. The average twist over the sequence is 36° ( 4°. The variation of the twist is similar to the one observed in d(CGCATATATGCG)2 (Yoon et al., 1988), showing in particular that the modification does not cause the helix to unwind. Comparison with Other Related Structures. Comparison of the d(5′-A4T5A6T7A8-3′):d(5′-T15A16X17A18T19-3′) part of the TAXA duplex with the central part of the duplex d(CGCATATATGCG)2 taken from the Brookhaven databank (Yoon et al., 1988; Yuan et al., 1992) shows that the stacking arrangements are almost identical for the central parts (Figure 7). The difference between the two amounts to an rms deviation of 2.63 Å for these five base pairs. However, the alternating pattern for the helical twist and rise through the alternating AT sequence are less pronounced for the TAXA sequence. Previously, several structures of araC-containing oligonucleotides (with a hydroxyl group in the arabino 2′position) have been determined (Schweitzer et al., 1994; Gao et al., 1991). These structures showed that the 2′OH group was positioned in the major groove, equivalent to what we observe in the case of the 2′-OCH3 in the TAXA oligonucleotide. In the X-ray structure of one of the araC-containing oligonucleotide duplexes it was found that the araC sugar pucker adopts an S-type conformation (Gao et al., 1991). An NMR structure of another araC oligonucleotide duplex shows the sugar pucker of araC to be of the N-type (Schweitzer et al., 1994), but with the overall duplex structure as B-DNA. Partly in agreement with that, the structural observations in the TAXA oligonucleotide imply that a normal B-type helix conformation is present and that the 2′-OCH3-araT


Gotfredsen et al.

Figure 8. Helical parameters for TAXA calculated using CURVES 3.1 and compared with canonical A-DNA (- - -) and B-DNA (- - -).

1H


adopts an S-type sugar conformation. A substitution on the 2′-oxygen in the arabino ring with longer alkyl chains would probably maintain the orientation and conformation of the sugar ring systems, retaining a B-form DNA configuration. This constitutes a possible path to create new antisense or antigene agents. When 2′-OCH3-araT is positioned in an oligonucleotide, it favors resistance to the 3′-exonuclease SV PDE, a dual DNA/RNA nuclease (Gotfredsen et al., 1994, 1996). This is an advantage over the normal DNA building blocks, which, when incorporated into a oligonucleotide, are easily degradable. The increased stability of TAXA may be caused by steric interference from the 2′-CH3O group in the arabino configuration so that the shape of the polymer does not fit into the active site of the enzyme. It has been reported that a 2′-CH3O group on the ribo face of the sugar ring does not interfere with the digestion by SV PDE (Sproat et al., 1989) so that the degradation is as fast as for a normal DNA strand. Molecular Design. The structural study in this work shows that 2′-OCH3-araT in the TAXA duplex adopts a B-like helix conformation with only a slightly lower DNA thermal stability and enhanced enzymatic stability toward SV PDE (Gotfredsen et al., 1994, 1996). The 2′OCH3 group of X17 is directed toward the major groove with the possibility of tethering a longer alkyl chain to that site without essentially disturbing the B-like conformation. RNA oligonucleotides modified with CH3O in the 2′position adopt N-type sugar conformations yielding a typical A-DNA structure (Lubini et al., 1994; Blommers et al., 1994). This is contrary to what is seen for TAXA, but in accordance with the suggestion that a strong electronegative group at C2′ determines the sugar pucker conformation for various sugar nucleosides (Uesugi et al., 1979; Guschlbauer and Jankowski, 1980). Thus, the 2′OCH3-araT adopts a C2′-endo conformation (S), implying that the electronegative -OCH3 in an arabino position will favor an S-type sugar conformation, whereas if the OCH3 is positioned in a 2′-ribo position, it will favor an N-type sugar conformation. Further studies on the ability of a 2′-O-alkylarabinonucleotide to enforce a B-DNA conformation when bound to an RNA strand would be of interest since the 2′-OCH3-RNA nucleotides can induce a local A-DNA-type helix when duplexed to a complementary DNA strand (Lubini et al., 1994). The observations described in this paper indicate that 2′-O-substituted araT-modified nucleotides are promising as alternatives to the normal deoxy nucleotides for use in antisense and antigene strategies. Furthermore, functionalization of the 2′-position of the deoxyribose ring in the arabino configuration with other groups, linker chains, and moieties such as intercalators and groove binders would yield molecules to test many interesting biological questions. ACKNOWLEDGMENT

We are grateful to Dr. F. Jensen and Dr. P. C. Stein for setting up some of the programs and for many helpful discussions. We thank Professor T. L. James and Dr. H. Liu, University of California, San Francisco, for providing the MARDIGRAS and the RANDMARDI programs and for helpful guidelines. We also thank The Danish Natural Science Research Council for generous financial support. LITERATURE CITED Arnott, S., and Hukins, D. W. L. (1972) Optimised Parameters for A-DNA and B-DNA. Biochem. Biophys. Res. Commun. 47, 1504-1510.

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BC960061F

Structure of a DNA Duplex Containing a Single 2 '-O-Methyl-Î²-d-araT

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