NMR Structure Determination of a Modified DNA Oligonucleotide

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Bioconjugate Chem. 2004, 15, 260−269

NMR Structure Determination of a Modified DNA Oligonucleotide Containing a New Intercalating Nucleic Acid Christina B. Nielsen,*,† Michael Petersen,† Erik B. Pedersen,† Poul Erik Hansen,‡ and Ulf B. Christensen† Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark, and Department of Life Science and Chemistry, University of Roskilde, P.O. Box 260, DK-4000, Denmark . Received October 22, 2003; Revised Manuscript Received February 3, 2004

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C1T2C3A4A5C6X7C8A9A10G11C12T13):d(A14G15C16T17-T18G19X20G21T22T23G24A25G26). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used twodimensional 1H NMR spectroscopy to determine a high-resolution solution structure of the weak INAINA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 Å. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.

INTRODUCTION

There has been a long search for new oligonucleotide analogues having different properties than natural DNA and RNA. This search has resulted in a very large selection of nucleotide analogues such as PNA (1), LNA (2), R-L-LNA (3), HNA (4), ANA (5), CNA (6), and CeNA (7). For a recent review see Leumann (8). Many of these analogues bind with improved affinity to DNA and/or RNA, but all of them bind even stronger to a complementary strand of the same type of analogue, i.e., they have a high affinity for self-hybridization. This is a large problem if you want to target both strands of a DNA duplex or a secondary structure of RNA like a hairpin (9, 10). To overcome this problem there has been an intensive search for analogues that bind preferentially to the target and not to the analogue itself, which has resulted in a few new analogues (11, 12). In this paper we present a novel intercalating nucleotide analogue (INA). The concept of intercalation was proposed in 1961 by Lerman (13) and have since been * To whom correspondence should be addressed. E-mail: [email protected]. † University of Southern Denmark. ‡ University of Roskilde. 1 Abbreviations: DQF-COSY, double quantum-filtered correlation spectroscopy; dsDNA, double-stranded DNA.; ssDNA, single-stranded DNA.; EDTA, ethylenediaminetetraacetic acid; INA, intercalating nucleic acid; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; rMD, restrained molecular dynamics; RMSD, root-mean-square deviation; ISPA, isolated spin pair approximation; TOCSY, total correlation spectroscopy.

subject for intense investigation (14). Some intercalators can be used as fluorescent labels and are applied universally in biology, biotechnology, and medicine (15-17). The pyrene moiety is one of the most heavily investigated molecules in terms of base stacking, thermal stabilization, and fluorescent abilities in DNA oligonucleotides (18-27). It has been suggested as an artificial nucleobase in DNA (28), attached to modified nucleotides such as LNA (29), and inserted in DNA with a glycerol backbone (30). These investigations showed a loss of stability upon hybridization except for the last example where a minor stabilization was observed when hybridized to DNA. Pyrene has also been tested as an intercalator covalently attached to the O2′ in a uracil nucleotide through a methylene linker. The investigations showed a promising stabilization when inserted in and hybridized to DNA whereas hybridization to RNA caused destabilization (31). Insertion in RNA caused destabilization when hybridized to both RNA and DNA (32). Recently, very promising results were presented, for a new intercalating nucleic acid (INA), consisting of a 1-O(1-pyrenylmethyl)glycerol (33, 34) (Scheme 1). These results showed that when INA is inserted as a bulge and hybridized to DNA, large thermal stabilizations are observed, whereas hybridization to RNA gives minor destabilizations. This makes INA highly promising as one of a few modifications that are able to discriminate between DNA and RNA. Fluorescent measurements show a decrease in fluorescence emission upon excitation at 340 nm after hybridization (33). This indicates that the pyrene moiety intercalates into the DNA double helix (27, 35).

10.1021/bc0341932 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/02/2004

Structure of an Intercalating Nucleic Acid in DNA Scheme 1. A Schematic Drawing of the Intercalating Pseudo Nucleotide (INA), with Numbering of Protons Included

Scheme 2. Numbering Scheme for the Four Duplexes Investigateda

a ”INA” indicates that an INA pesudo nucleotide has been incorporated and “-” indicates a normal DNA strand with no insertions.

In the present investigation, the objective was to explore the structural implications of two INAs placed directly opposite each in a dsDNA duplex and to determine whether INAs possess highest affinity for selfhybridization or for hybridization to complementary DNA. EXPERIMENTAL PROCEDURES

Sample Preparation. Samples of duplex 1 and 4 were prepared for NMR investigations (Scheme 2). The two modified strands were synthesized as described elsewhere (33). The unmodified oligonucleotides were purchased from DNA Technology, A° rhus, Denmark. The samples were made by dissolving the two single strands in equivalent amounts in 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, and 0.05 mM EDTA. The samples were heated to 80 °C and slowly cooled to achieve hybridization. The final concentrations of the duplexes were 1.9 mM. For experiments in D2O, the solid sample was lyophilized three times from D2O and redissolved in 99.96% D2O (Cambridge Isotope Laboratories). For experiments involving exchanging protons, the sample was dissolved in 90% H2O and 10% D2O. The numbering scheme used for the duplexes is shown in Scheme 2. Thermal Stability Studies. The thermal stability was measured of duplexes 1-4 (Scheme 2). The thermal stability was determined spectrophotometrically on a Perkin-Elmer spectrophotometer equipped with a thermoregulated Peltier element. Hybridization mixtures were prepared by dissolving equimolar amounts (2.53.5 µM) of the oligonucleotides in 10 mM Na2HPO4‚H2O (pH 7.0), 140 mM NaCl, and 1 mM EDTA. Measurements were done at pH ranging from 4 to 10 in steps of one unit. The pH was regulated using NaOH and HCl. The melting temperature was monitored while the temperature was changed linearly from 20 to 70 °C (1 °C min-1) and then back to 20 °C. The melting temperatures were obtained as the average between the maxima/minima of the first derivatives of the melting curves. NMR Experiments. The NMR experiments were performed on a Varian INOVA 600 spectrometer at 25

Bioconjugate Chem., Vol. 15, No. 2, 2004 261

°C unless otherwise stated. For duplex 1, a NOESY spectrum in D2O was acquired using 2048 complex points in t2 and a spectral width of 6000 Hz. A total of 512 t1 experiments were recorded using the States phase cycling scheme. The residual signal of HOD was removed by lowpower presaturation. A NOESY spectrum in H2O was obtained using the watergate pulse sequence. The spectrum was acquired with 2048 complex points in t2 and a spectral width of 12000 Hz. A total of 512 t1 experiments were recorded using the States phase cycling. A DQFCOSY spectrum and TOCSY spectra with mixing times of 30 and 70 ms were obtained using the States phase cycling scheme. For duplex 4, two NOE buildup curves were obtained, one in D2O and one in H2O. In D2O, the NOESY spectra were acquired using 2048 complex points in t2 and a spectral width of 6000 Hz. A total of 512 t1 experiments were recorded using the States phase cycling scheme. The NOESY spectra with mixing times of 50, 100, 150, and 200 ms were obtained sequentially without removing the sample from the magnet. 64 scans were acquired for each t1 value with a repetition delay of 4.0 s between each scan. The residual signal of HOD was suppressed by lowpower presaturation. In H2O, the watergate-NOESY spectra were acquired using 2048 complex points in t2 and a spectral width of 12000 Hz. A total of 680 t1 experiments were recorded using the States phase cycling scheme. Spectra with mixing times of 100, 200, and 300 ms were obtained sequentially without removing the sample from the magnet. 64 scans were acquired for each t1 value with a repetition delay of 2.0 s between each scan. A DQF-COSY spectrum and TOCSY spectra with mixing times of 30 and 70 ms were obtained using the States phase cycling scheme. An inversion recovery experiment was recorded to estimate the T1 relaxations rates. All the acquired data were processed using FELIX (Version 98, MSI, San Diego, CA). All spectra were apodized by a skewed sinebell squared in F1 and F2. The four spectra used in the D2O NOE build up were linear predicted in F1 from 340 up to 400 points and baselinecorrected in F2 using the Flatt algorithm. The three spectra used in the build up in H2O were linear-predicted in F1 from 420 up to 600 points and baseline corrected using the Flatt algorithm in F2. Distance Restraints. The upper and lower diagonal part of each of the four NOESY buildup spectra in D2O were separately integrated with FELIX, yielding a total of eight peak intensity sets which were corrected for minor saturation effects using the T1 relaxations rates determined. A modified ISPA approach, as suggested by Wijmenga and Van Buuren, was used to calculate interproton distance bounds from the eight intensity sets (36). Bound widths were set to (10% of the distances obtained. The same procedure was used for the buildup spectra in H2O, but here the bounds were set to (15% of the distances. Base planarity restraints and backbone torsion angle restraints were added for the two terminal base pairs: C1:G26, T2:A25, C12:G15, and T13:A14. The values of the 27 backbone angles included were: -90° < R < -30°, -210 < β < -150°, 30° < γ < 90°, 150° < < 210°, and -150 < ζ < -90°. These values encompass both A- and B-type duplex structures. Restrained molecular Dynamics. Two starting structure for the structure calculations were generated: an A-type and a B-type duplex. These were built with the nucgen and nukit modules of AMBER 6 (37). The INA nucleotides were added using the xleap module and

262 Bioconjugate Chem., Vol. 15, No. 2, 2004

Nielsen et al.

Table 1. Melting Temperatures of INA and DNA Duplexes Measured at Different pH Valuesa strand duplex 1 2 3 4

1

pH

2

4.2

5.0

6.1

7.0

8.0

9.0

10.0

DNA DNA 30.6 43.6 47.8 47.2 DNA INA 49.7 54.7 54.5 INA DNA 45.4 51.9 52.5 INA INA 36.1 46.0 46.5

49.1 55.7 54.7 48.5

49.6 54.1 52.1 46.5

43.3 51.1 46.4 40.9

a The measurements were done in 1 mM EDTA, 10 mM Na2HPO4‚H2O, and 140 mM NaCl.

atomic charges for the INA nucleotides were calculated using the RESP procedure as described by Bayly et al., (38). All distance restraints were incorporated into an rMD procedure for structure refinement. 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 final derivative of 0.01 Å. A distance dependent dielectric constant ( ) r) was employed to account for solvent effects. The pseudo-energy term used to enforce the distance restraints was:

{

k1(r - r1)2 when r1 > r when r2 g r e r1 ENOE ) 0 k2(r - r2)2 when r2 > r r1 and r2 are the lower and upper distance bounds, and k1 and k2 are the force constants. A force constant of 50 kcal mol-1 Å-2 for lower and upper bounds was assigned to all distance restraints. Helix parameters were calculated with the program CURVES 5.2 (39, 40). RESULTS

Thermal Stability. The thermal stabilities of the four duplexes shown in Scheme 2 were determined and compared (Table 1). Sharp monophasic transitions were obtained with hypochromicities of 1.1-1.2. No indications of biphasic transitions were detected. The measurements were performed at different pH values to investigate the pH dependency of INAs thermostability, and it can be noted that both DNA and INA duplexes are most stable in slightly basic environments. Incorporation of one INA modification in either of the strands (duplex 2 and 3) creates an increase in melting temperature of up to 6.6 °C. The difference in melting temperature reveals a small sequence dependency of the stabilization created by INA. Incorporation of INA modifications in both strands (duplex 4) creates a decrease in melting temperature of up to 7.2 °C compared to the duplexes with INA in one only of the strands (duplexes 2 and 3) and 0.6 °C compared to the unmodified duplex (duplex 1). These results suggest that INA does not have a high affinity for self-hybridization; on the contrary, insertion of two INAs directly opposite each other creates a significant thermal destabilization of the duplex compared to when one INA is inserted in one of the strands. The degree of destabilization when INA modifications are inserted opposite each other is increasing when going to acidic pH values. Spectral Analysis. The 1D 1H NMR spectrum of the INA:INA duplex exhibits only lines from the expected duplex, and there were no signs of alternative hybridizations. The NOESY spectra of the INA:INA duplex exhibit the characteristic features of right-handed dsDNA se-

quential connectivities. The assignment of the nonexchangeable DNA protons in the INA:INA duplex was performed using standard methods (41-44). Both aromatic (H6, H8, and H2) and sugar protons (H1′, H2′, H2′′, H3′, H4′, H5′, and H5′′) were assigned. The NOESY spectrum recorded with short mixing time (50 ms) allowed unambiguous assignments of the H2′ and H2′′ resonances. The H6/H8-H1′ cross-peaks were all weak, suggesting that the glycosidic angle is in the antiposition. The assignment of the exchangeable protons was obtained from the NOESY spectra in H2O (45). The chemical shift values and the NOE connectivity pattern of the imino protons observed were in accordance with normal Watson-Crick base pairing. The unmodified duplex was assigned using the same strategy as for the INA:INA duplex; all determined chemical shift values of the unmodified DNA duplex indicated a canonical B-type helix structure. A selection of the chemical shift values of the DNA nucleotides in the INA:INA duplex are given in Table 2 and compared to the corresponding values of the unmodified duplex. The assignment of the INA protons was performed using NOESY, DQF-COSY, and TOCSY spectra. The NOESY spectra showed that both INA nucleotides are intercalated, resulting in a large number of NOEs involving both of the aliphatic linkers and the pyrene moieties. The lines arising from INA and from the neighboring nucleotides are broader than other DNA lines, indicating more dynamics in the insertion area. The chemical shift values of the INA intercalators are given in Table 3. Structure Calculations. More than 1000 NOE crosspeaks were observed in the NOESY spectra obtained with a 200 ms mixing time in D2O. The accuracy of the integration of some cross-peaks was hampered by spectral overlap. These cross-peaks were not included in the modified ISPA calculations. The integration of the crosspeaks in the four buildup spectra was done separately for each side of the diagonal, resulting in two times four sets of 406 and 415 NOE cross-peaks intensities, respectively. This gives a total of 3284 integrated cross-peaks intensities used in the modified ISPA calculation. Crosspeak intensities that correspond to covalently fixed distances in the dsDNA were used for internal calibration and were therefore not converted into distance restraints. As the number of covalently fixed distances in DNA is rather limited, some internal sugar distances that are fairly independent of the sugar conformation were used for calibration as well. There are 19 internal protonproton distances in a deoxyribose; these distances were determined at five different pseudorotation angles in the area of the two low energy conformations C3′-endo and C2′endo. In each of the 10 sets, NOESY cross-peaks corresponding to 77 deoxyribose distances and 19 covalently fixed distances were observed and thus yielded 96 distances used in calibration of unknown distances. The final bounds used in the rMD calculations were the average distances from the 10 distance sets ( 10%. The calculations returned 443 interproton distances; of those, 39 involved an INA proton. Evaluation of the NOESY spectra in H2O showed normal Watson-Crick bonding in the DNA parts of the duplex except between A9:T18, justifying inclusion of 28 hydrogen bond distance restraints. Three hydrogen bonds were included for each of the six GC base pairs and two hydrogen bonds for each of the five AT base pairs with upper and lower bounds of 1.74 and 2.10 Å, respectively. From the NOESY buildup in H2O, a total of 36 distance restraints were determined using the same method as in D2O. But here only covalent fixed distances were used

Structure of an Intercalating Nucleic Acid in DNA


Table 2. A Selection of Chemical Shifts (in ppm) for the DNA Nucleotides in the INA:INA Duplexa C1 T2 C3 A4 A5 C6 X7 C8 A9 A10 G11 C12 T13 A14 G15 C16 T17 T18 G19 X20 G21 T22 T23 G24 A25 G26

H1′

H2′

H2′′

H3′

H6/H8

H5/H2/CH3

5.86 (5.91) 6.12 (6.15) 5.34 (5.43) 5.72 (5.87) 5.89 (6.10) 5.49 (5.71)

2.24 (2.25) 2.19 (2.22) 1.98 (2.01) 2.70 (2.78) 2.39 (2.61) 2.02 (1.91)

2.57 (2.60) 2.52 (2.57) 2.26 (2.32) 2.77 (2.89) 2.60 (2.83) 2.14 (2.35)

4.64 (4.68) 4.88 (4.90) 4.82 (4.85) 5.01 (5.06) 4.98 (5.00 4.85 (4.70)

7.88 (7.92) 7.59 (7.61) 7.48 (7.51) 8.16 (8.26) 7.92 (8.11) 6.80 (7.16)

5.95 (6.00) 1.66 (1.70) 5.64 (5.74) 7.15 (7.24) 7.36 (7.63) 4.82 (5.15)

5.13 (5.37) 5.63 (5.82) 5.93 (5.98) 5.69 (5.74) 6.02 (6.06) 6.24 (6.26) 5.97 (5.99) 5.84 (5.98) 6.01 (6.04) 5.98 (6.08) 5.51 (5.81) 5.20 (5.70)

1.97 (1.92) 2.75 (2.73) 2.61 (2.62) 2.44 (2.45) 2.16 (2.20) 2.28 (2.30) 2.47 (2.64) 2.69 (2.22) 2.07 (1.65) 2.02 (2.15) 1.48 (2.06) 2.52 (2.71)

2.15 (2.28) 2.79 (2.87) 2.79 (2.81) 2.58 (2.60) 2.44 (2.46) 2.28 (2.30) 2.63 (2.81) 2.69 (2.45) 2.51 (2.57) 2.41 (2.58) 2.84 (2.46) 2.60 (2.75)

4.72 (4.89) 5.02 (5.03) 5.01 (5.04) 4.91 (4.92) (4.75) 4.54 (4.56) 4.83 (4.84) 4.98 (-) 4.74 (4.65) 4.82 (-) 4.72 (4.89) 4.97 (5.00)

7.55 (7.37) 8.19 (8.20) 8.03 (8.05) 7.56 (7.57) 7.39 (7.42) 7.55 (7.59) 8.00 (8.01) 7.87 (7.57) 7.43 (7.36) 7.37 (7.46) 7.00 (7.31) 7.48 (7.86)

5.75 (5.48) 7.00 (7.22) 7.45 (7.50)

5.91 (5.95) 5.67 (6.00) 5.59 (5.75) 5.37 (5.43) 6.09 (6.12) 5.97 (6.01)

2.77 (2.54) 1.96 (2.06) 1.87 (1.96) 2.69 (2.71) 2.63 (2.66) 2.24 (2.39)

2.77 (2.75) 2.28 (2.54) 2.24 (2.34) 2.69 (2.71) 2.88 (2.90) 2.37 (2.39)

5.00 (4.90) 4.72 (4.85) 4.76 (4.86) 4.96 (4.99) 5.01 (5.03) 4.60 (4.62)

8.07 (7.64) 7.16 (7.24) 7.16 (7.27) 7.86 (7.91) 8.08 (8.12) 7.61 (7.64)

H1/H3

H4

H4

8.53 (8.61) 7.73b (-) 7.18b (-) 6.82 (8.05)

6.80 (6.84) 6.28b (-) 5.59b (-) 5.86 (6.39)

7.48 (8.42) (-) (-)

6.36 (6.58) (-) (-)

8.16 (8.20)

6.51 (6.51)

(-)

(-)

8.01 (8.05)

6.46 (6.51)

7.95b (-)

6.04b (-)

13.82 (13.86)

12.72 (12.77) 5.32 (5.33) 1.71 (1.74) 7.92 (-)

(-) 12.80 (12.83)

5.30 (5.25) 1.57 (1.61) 1.58 (1.69)

1.30 (1.29) 1.58 (1.66)

13.90 (14.06) (13.82) 11.00b (12.61) 10.61 (12.77) 13.33 (13.82) 13.71 (13.80) 12.54 (12.59)

7.73 (7.78) (-)

a The values of the unmodified duplex are given in parentheses. The values are measured at 25 °C relative to water unless otherwise stated. b Measured at 5 °C relative to water.

Table 3. Chemical Shift Values (ppm) of the INA Nucleotidesa H2 H3 H4 H5 H6 H7 H8 H9 H10 H1′1/H1′2 H2′ H3′1/H3′2 H4′1/H4′2 a

X7

X20

6.67 6.93 7.30 7.38 7.37 7.22 7.14 7.58 7.55 4.09/4.16 4.47 3.79/3.84 4.84/4.84

7.21 6.98 7.14 7.20 7.07 6.81 6.72 7.53 7.36 4.20/4.29 4.61 3.86/3.98 4.30/4.54

The values are given at 25 °C relative to water.

for calibration, and bounds were set to the average distances (15%. None of the restraints from the spectra acquired in H2O involved an INA proton. Thus, a total

Scheme 3 Dispersion of the Distance Restraints Used in the RMD Calculationsa

a Dashed lines are distances derived from NOESY spectra in H2O; solid lines are distances from NOESY spectra in both D2O and H2O.

of 479 interproton restraints were used in the rMD calculations. The dispersion of the distance restraints used in the rMD calculation is shown in Scheme 3. Twenty final structures starting from both A- and B-type starting structures, respectively, were generated. All the structures converged to one family of conformations, but the RMSD between structures generated from A- and B-types were slightly larger than internally in each of the two families. Since no A-type properties are


Nielsen et al.

tures all converged to the same family of structures as with the force constants set to 50 kcal mol-1 Å-2. The structure obtained is not strongly dependent on the exclusion of any of the restraints. In particular, calculations performed without the Watson-Crick restraints yielded the same structure but with a slightly larger RMSD. Helix Parameters. Helical parameters for the 20 final structures were analyzed with the program CURVES 5.2 (39, 40). Plots of some global helical parameters for the INA:INA duplex are shown in Figure 5. The coordinates for the twenty final structures are deposited in the RSCB Protein Data Bank under pdb ID 1S88 (46). Figure 1. Four different views of 20 superimposed structures. (A) All residues are superimposed. (B) Residues [1-6;21-26] are superimposed. (C) Residues [8-13;14-19] are superimposed. (D) Residues [5-9;18-21] are superimposed. Hydrogens have been omitted for clarity.

observed in the final structure, only the 20 structures generated from the B-type structure are further analyzed. Four different views of the duplex are shown in Figure 1, with the RMSD for each ensemble indicated. The average all atom RMSD of the coordinates of the 20 B-type structures was 1.95 Å (Figure 1A). This rather high value, along with line broadening of the INA protons as well as the protons from the neighboring nucleotides, reflects the dynamics of the duplex. Segments of the duplex all had RMSDs below 1.0 Å (Figure 1B-D). The average sum of violations was 12.9 Å for the B-type starting structure. Just eight violations greater than 0.2 Å were observed and none greater than 0.3 Å. The reliability of the structure calculation was carefully evaluated. rMD calculations were performed with distance restraints force constants changed to either 25 or 100 kcal mol-1 Å-2, respectively. The generated struc-

DISCUSSION

Spectral Data. The NOESY cross-peaks shows that the duplex has a right-handed helix conformation with all nucleotides in the anti conformation. The sequential pathway is broken at the INA modifications due to their unusual backbone. The H6/H8-H1′ region of the 200 ms NOSEY spectrum is shown in Figure 2. It shows some of the sequential connectivities, and examples of INADNA cross-peaks. The chemical shift values displayed in Table 2 demonstrate a close agreement between the modified and the unmodified duplex except for the protons near the INA modifications. The differences in chemical shift are primarily displayed at C6, C8, T18, G19, and G21. The changes in chemical shift can largely be explained by enhanced ring current effects induced by the pyrene moieties. It is difficult to make a pattern in the changes in chemical shifts observed, but it seems as if the nucleotides on the 5′-site of the intercalators (G6 and G19) obtains an upfield shift whereas the nucleotides on the 3′-site (C8 and G21) shift downfield. The chemical shifts of the intercalators are rather difficult to discuss,

Figure 2. A section of the 200 ms NOESY spectrum displaying the H6/H8-H1′ region. The sequential connectivities for the C1T13 strand are shown. Note that the connectivity is broken between C6 and C8, due to the missing sugar ring in INA. Cross-peaks between the DNA protons C6:H1′ and C19:H1′ and some of the aromatic protons on the intercalators, X7 and X20, are shown. Cytosine H6-H5 cross-peaks are marked with an asterisk. The C6:H6-C6:H5 cross-peak is not present, due to an upfield shift of C6:H5 induced by the pyrene moiety of X20.


but compared to pyrene, the aromatic protons in the pyrene moieties are shifted approximately 1 ppm upfield. This is probably due to ring current effects from one INA to the other and vice versa, indicating that both pyrene moieties are intercalated. These findings are supported by calculations of ring current effects, which display a similar trend (47, 48) The imino protons of G19 and G21 are shifted upfield 1.61 and 2.16 ppm, respectively. Such a dramatic upfield shift has been observed before in the literature for imino protons in guanines stacking with pyrenyl ring systems (21). Upfield shift of imino protons can be caused by two things: imino protons, with sharp and narrow lines, shifted upfield indicate significant ring current effects due to stacking interactions, whereas, imino protons,with broad lines due to rapid exchange, indicate disruption of the base pair hydrogen bonding (21). The reason for the dramatic upfield shift observed for G19:H1 and G21:H1 are probably a combination of the two. They have broad lines and G21:H1 can only be observed in the NOESY spectrum in H2O at 5 °C, but the characteristic crosspeaks defining Watson-Crick hydrogen bonding are present in the spectra, indicating that base pairing does occur between G19:C8 and G21:C6. The broad lines are presumably due to internal motion rather than disruption of the base paring. It is interesting that the chemical shift changes observed at T18 are greater than at G19, especially T18: H2′ is shifted 0.58 ppm upfield. Also no Watson-Crick base pairing is observed in the base pair A9:T18. This could be due to necessary structural compensations induced by the intercalation of the INA modfications. The reason it occurs at the A9:T18 base pair rather than at one of the flanking G:C base pairs is that A:T base pairs are more prone to disruption than G:C base pairs. This could explain why the chemical shift changes at T18 are greater than at G19, even though G19 is a neighbor to the intercalator ×20. Description of the Structure. Qualitatively, the NOESY spectra of the INA:INA duplex indicate that it adopts a right-handed B-type helix conformation, with all the bases in the anti conformation and all forming normal Watson-Crick base pairs. However, Figure 3A reveals that even though the ends of the duplex looks like normal right-handed B-type helixes, some major changes occur in the area of the inserted intercalators. One obvious consequence of the intercalators is a dramatic unwinding of the helix in the insertion area creating a ladderlike structure. Unwinding of the helix as a consequence of intercalation is a common phenomenon for noncovalent bound intercalators (49). However, in this case where the INA nucleotide provides its own backbone, unwinding was not necessarily expected. INA is designed to intercalate in the opposite strand, but Figure 4 reveals that when two INA are inserted opposite to each other they prefer to stack with each other, and X7 is therefore intercalated in the opposite strand as expected whereas X20 intercalates more or less in the strand where it is inserted. Thus both pyrene moieties stack in the same DNA strand, creating good stacking between G19, X7, X20, and G21, whereas stacking with C6 and C8 is less predominant. This shows that INA prefers to stack with G rather than C, due to the larger aromatic system of guanine. Due to the difference in intercalation, the conformation of the linker of the two INA nucleotides is different. The linker of X7 is quite extended whereas the linker of X20 is rather compact. Figure 4 shows that both intercalators are oriented the same way in the duplex relative to the


Figure 3. (A) Space-filling representations of the structure viewed from the minor groove (left) and turned 90° (right). (B) Stick representations of the structure. The helix axis calculated with CURVES is shown, and a ribbon has been added to emphasize the backbone. Hydrogens have been omitted for clarity.

Figure 4. Top: Base pair stacking in the central part of the INA:INA duplex viewed from the major groove. Bottom: Same picture viewed along the helix axis, the backbone is not shown. There seems to be good stacking between G19, X7, X20, and G21, whereas the stacking of the pyrenes with C6 and C8 is rather limited.

strand direction. This could indicate that the pyrene moieties of the INA nucleotides have a preferred orientation when they intercalate. Dynamic Considerations. The structure of the INA: INA duplex (duplex 4) shows that both pyrene moieties are intercalated. This observation is supported by chemical shift values observed for the protons in the pyrene moiety. They are shifted approximately 1 ppm compared to pyrene, indicating ring current effects from one pyrene


Nielsen et al.

Figure 5. A selection of the helix parameters calculated for the INA:INA duplex. The parameters were calculated using CURVES 5.2 and are compared to values of canonical A-type duplex (- - -) and B-type duplex (- - -) parameters. No values are calculated for the intercalators X7 and X20.

moiety affecting the other and vice versa. However, all INA protons, as well as the protons from the neighboring nucleotides, have line widths broader than normal, indicating intermediate dynamics in this area. These dynamics have to occur on a fast time scale since only one set of peaks can be observed in any of the acquired spectra. The observed dynamics of the duplex could have its origin in several different patterns of movement by the pyrenes. They could be intercalated alternately and only briefly be intercalated both at the same time, they could simultaneously move in and out of the helix, or they could be sliding on top of each other either sideways or back and forth. Considering the amount of cross-peaks identified between X7 and X20, the two first models do not seem likely. If the pyrenes only were stacking with each other for a short time, the number of cross-peaks and their intensities would probably be lower. To distinguish between the two other possibilities is more problematic, but since the minor groove has been shown to be extensively widened in the duplex, the last option where the two pyrene moieties are sliding back and forth does seem more appealing. Unfortunatly, no definitive answer can be given. Helix Parameters. There are three major categories of helical parameters: axis-base pair, intrabase pair and interbase pair parameters (50). For the INA:INA duplex some of these parameters are shown in Figure 5. However, no data are available for the intercalators, and the data for the terminal base pairs are probably less reliable due to dynamic processes and a lower density of restraints and are only included for completeness. The global helix axis is fairly straight (Figure 3B), but it should be noted that the axis does not pass through the intercalators, as they seem to be shifted toward the minor groove. The width of the minor groove, measured as the shortest interstrand phosphorus distance (reduced by 5.8 Å to account for the van der Waals radius of two phosphate groups), is reported in Figure 6. For classical B-type helices, a value of approximately 5.7 Å is expected (51), but in the INA structure, a remarkable widening of the minor groove is observed throughout the duplex. The widening is most dramatic in the insertion area where enlargements up to 10.1 Å are observed. This remarkable

Figure 6. The minor groove width measured as the smallest distance between two interstrand phosphorus atoms reduced by 5.8 Å to account for the van der Waals radius of two phosphate groups. A greatly enlarged minor groove width in the central part of the helix is observed.

result indicates that even though the two INAs seemingly are stacking well, they also push the two strands of the duplex apart and hence have a pronounced influence on the minor groove width of the entire duplex. The calculated values of the intrabase pair parameters shear, buckle, and propeller twist all deviate from normal A- and B-type DNA in the central part of the helix. Changes in buckle have been reported earlier to be connected to changes in the sugar conformations (52, 53), but values of the pseudorotation angle show a majority of S-type conformation in all nucleotides except C6 (results not shown). Even though propeller twist for structures of oligonucleotides determined by NMR normally are more negative than for canonical B-type helixes


(54-57), the large negative value for the A9:T18 base pair is noteworthy. Together with the large positive buckle, it explains why no Watson-Crick hydrogen bonding could be observed for this base pair. The unusually large positive values of buckle and the large changes in shear, combined with the large changes in the interbase pair parameter tilt in the insertion area, demonstrate the dramatic adaptations the structure has to perform due to the insertion of the two INAs. Stability of INA Duplexes. The thermal stability measurements displayed in Table 1 shows that one INA nucleotide inserted in either of the strands increases the affinity for the complementary ssDNA. This increase in melting temperature was expected from previous results (33). We have shown that even though INA has the very high affinity for its target DNA, it has a lower affinity for complementary INA, when the two INA insertions are placed opposite each other. By adjusting pH to 5.0 (see Table 1), it is possible to optimize the difference in thermal stability between the INA:DNA hybrid and the INA:INA duplex to 13.6 °C. However, thermal stability measurements of duplexes with INA molecules inserted in each strand, displaced with one to three base pairs, show a stabilizing effect of up to 7.0 °C, indicating that INA in general have a high affinity for other sequences with INA inserted (unpublished results). Even though the intercalation of the two pyrene moieties makes a severe pertubation to the duplex structure, the thermostability of the INA:INA duplex is only slightly lower than that of the unmodified dsDNA duplex. One possible explanation of this could be that even though two intercalators inserted directly opposite each other distort the duplex, they also create an area with an increased flexibility due to the greater mobility of the backbone of the intercalators compared to a normal nucleotide. This enables the duplex to make major changes in the insertion area without having to rearrange the rest of the duplex, thereby creating independent helixes on each side of the intercalators. Another factor minimizing the degree of destabilization is the strong stacking of the INAs, contributing to the stability of the duplex, an effect observed previously with bipyridyl C-nucleotides (58). This enhanced stacking in the duplex induced by the pyrene moieties is probably the reason insertion of INAs displaced by one to three base pairs stabilizes the duplex. Such an INA arrangement would intuitively destabilize the duplex even further because the structural adaptations necessary to fit both intercalators in the helix would spread over a greater part of in the duplex. However, due to the enhanced stacking abilities of the pyrene moieties compared to the natural nucleobases, insertion of INAs creates an enhanced stacking in the duplex in a larger area, thereby stabilizing the duplex. One of the conclusions obtained in this investigation is that insertion of two INA intercalators opposite each other creates a minor thermal destabilization of the duplex. This destabilization is founded in an unwinding of the helix in the insertion area, where the pyrene moieties exhibit good stacking abilities but also push the two strands apart, creating an extensively widened minor groove. Thermal stability measurements suggest that the destabilizing effect of inserting INA in both strands is specific for the case where they are inserted directly opposite each other; otherwise insertion of INA in both strands have a stabilizing effect. The measurements also show that by adjusting pH it is possible to optimize INA affinity for a complementary DNA target over a complementary INA target.


Intercalating nucleic acid is thus one of the first nucleotide analogues that have reduced affinity for hybridization to a complementary strand of its own kind, while still processing a highly increased affinity for hybridizing to its complementary DNA strands, making targeting of both of the complementary strands of a DNA duplex possible. On the basis of the high-affinity, high specificity and low self-affinity, we believe that INA can be used with great advantage in a versatile range of applications where hybridization to single- as well as double-stranded targets is desired. ACKNOWLEDGMENT

We would like to thank Professor Michael P. Williamson at the University of Sheffield for calculating the ring current values for the duplexes. We thank the Danish National Research Foundation, The Danish Natural Science Research Foundation, and the Danish Technical Research Foundation for financial support. LITERATURE CITED (1) Nielsen, P. E., and Haaima, G. (1997) Peptide nucleic acid (PNA). A DNA mimic with a pseudopeptide backbone. Chem. Soc. Rev. 26, 73-78. (2) Koshkin, A. A., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., Olsen, C. E., and Wengel, J. (1998) LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition. Tetrahedron 54, 3607-3630. (3) Rajwanshi, V. K., Hakansson, A. E., Dahl, B. M., and Wengel, J. (1999) LNA stereoisomers: xylo-LNA (beta-D-xylo configured locked nucleic acid) and alpha-L-LNA (alpha-Lribo configured locked nucleic acid). Chem. Commun. 13951396. (4) Van Aerschot, A., Verheggen, I., Hendrix, C., and Herdewijn, P. (1995) 1,5-Anhydrohexitol Nucleic-Acids, A New Promising Antisense Construct. Angew. Chem., Int. Ed. Engl. 34, 13381339. (5) Allart, B., Khan, K., Rosemeyer, H., Schepers, G., Hendrix, C., Rothenbacher, K., Seela, F., Van Aerschot, A., and Herdewijn, P. (1999) D-Altritol nucleic acids (ANA): Hybridisation properties, stability, and initial structural analysis. Chem.-Eur. J. 5, 2424-2431. (6) Maurinsh, Y., Rosemeyer, H., Esnouf, R., Medvedovici, A., Wang, J., Ceulemans, G., Lescrinier, E., Hendrix, C., Busson, R., Sandra, P., Seela, F., Van Aerschot, A., and Herdewijn, P. (1999) Synthesis and pairing properties of oligonucleotides containing 3-hydroxy-4-hydroxymethyl-1-cyclohexanyl nucleosides. Chem.-A Eur. J. 5, 2139-2150. (7) Wang, J., Verbeure, B., Luyten, I., Lescrinier, E., Froeyen, M., Hendrix, C., Rosemeyer, H., Seela, F., Van Aerschot, A., and Herdewijn, P. (2000) Cyclohexene nucleic acids (CeNA): Serum stable oligonucleotides that activate RNase H and increase duplex stability with complementary RNA. J. Am. Chem. Soc. 122, 8595-8602. (8) Leumann, C. J. (2002) DNA analogues: from supramolecular principles to biological properties. Bioorg. Med. Chem. 10, 841-854. (9) Cheng, S., Van Houten, B., Gamper, H. B., Sancar, A., and Hearst, J. E. (1988) Use of psoralen-modified oligonucleotides to trap three-stranded RecA-DNA complexes and repair of these cross-linked complexes by ABC excinuclease. J. Biol. Chem. 263, 15110-15117. (10) Lohse, J., Dahl, O., and Nielsen, P. E. (1999) Double duplex invasion by peptide nucleic acid: a general principle for sequence-specific targeting of double-stranded DNA. Proc. Natl. Acad. Sci. U.S.A 96, 11804-11808. (11) Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., Jr., and Gamper, H. B., Jr. (1996) Oligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agents. Biochemistry 35, 11170-11176.

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BC0341932

NMR Structure Determination of a Modified DNA Oligonucleotide

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