NMR Studies of Fully Modified Locked Nucleic Acid (LNA) Hybrids

May 4, 2004 - LNA is a bicyclic nucleic acid analogue that contains one or more 2′-O,4′-C methylene linkage(s), which effectively locks the furano...
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Bioconjugate Chem. 2004, 15, 449−457

449

NMR Studies of Fully Modified Locked Nucleic Acid (LNA) Hybrids: Solution Structure of an LNA:RNA Hybrid and Characterization of an LNA:DNA Hybrid Katrine E. Nielsen, Jill Rasmussen, Ravindra Kumar, Jesper Wengel, Jens Peter Jacobsen, and Michael Petersen* Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, 5230 Odense M, Denmark . Received August 22, 2003; Revised Manuscript Received December 17, 2003

LNA is a bicyclic nucleic acid analogue that contains one or more 2′-O,4′-C methylene linkage(s), which effectively locks the furanose ring in a C3′-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.

INTRODUCTION

High-affinity binding to complementary nucleic acids has a number of applications, for example in oligonucleotide-based therapeutics and in the general area of biotechnology. From the vast number of chemically modified nucleotide analogues synthesized, it appears that a sugar engineered into an N-type (RNA-like) pucker usually conveys an increase in helical thermostability when hybridized with complementary RNA (1). Prominent examples of such N-type nucleic acid analogues are 2′-O-alkylated RNA (2), 2′F-RNA (3), phosphoramidates (4), HNA (5), and LNA (6-9). In LNA (see Figure 1), the furanose conformation is chemically locked in an N-type (C3′-endo) conformation by the introduction of a 2′-O,4′-C methylene linkage. LNAs1 have shown hitherto unprecedented thermal affinities when hybridized with either DNA (∆Tm ) 1-8 °C per modification) (6-11), RNA (∆Tm ) 2-10 °C per modification) (6-9, 11-13) or LNA (∆Tm > 5 °C per modification) (14). The high thermal stability of LNA modified nucleic acids is achieved with sequence specificity comparable to or slightly better than native nucleic acids (6). Thus, short, fully modified LNA oligonucleotides combine high-affinity binding toward DNA and RNA with minimal oligonucleotide length. LNA is emerging as a useful tool in therapeutics and diagnostics with an increasing number of reports appearing. In the context of antisense therapeutics, LNA/ DNA gapmers are recognized and cleaved by RNase H (13). Thus, the high-affinity binding properties of LNA can be employed to access mRNA targets with highly * To whom correspondence should be addressed. E-mail: [email protected]. † Odense University. ‡ University of Copenhagen. 1 We have defined LNA as an oligonucleotide containing one or more 2′-O,4′-C-methylene-β-D-ribofuranosyl nucleotide monomer(s).

Figure 1. Structure of LNA with torsion angles labeled (left) and stick plot showing the C3′-endo furanose conformation (right).

ordered tertiary structures. In a broader therapeutical view, LNAs have been employed as aptamers for transcription factor κB (NF-κB) (15) and have been successfully incorporated into DNAzymes (16). For use in diagnostics, LNA probes are employed for the detection of single nucleotide polymorphisms (17, 18). Reference 9 is a recent review on applications of LNA oligonucleotides. In this study, we have characterized an LNA:DNA hybrid and determined the solution structure of an LNA: RNA hybrid. In both of these hybrids the LNA strand is composed entirely of LNA monomers. This is the first structural study of fully modified LNA oligonucleotides. The base composition and numbering of the two hybrids is shown in Scheme 1. As for the LNA:RNA hybrid, we have previously studied the corresponding, isosequential unmodified DNA:RNA hybrid and the two partly modified hybrids with LNA modifications at position 5 and positions 2, 5, and 7, respectively (these hybrids are labeled LNA1:RNA and LNA3:RNA henceforth) (12, 19). These studies showed that with an increasing number of LNA nucleotides incorporated into the hybrids, an increasing A-like character of the duplex structure ensued. This is due to the LNA nucleotides being locked in a C3′-endo confor-

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Scheme 1. Numbering Scheme and Base Composition of the Two Hybrids Studieda

Figure 2. The distribution of the NOE restraints. The numbers of intranucleotide, sequential and cross-strand restraints are indicated. In addition to the restraints shown, six further restraints were also obtained (C1-G3, T7-C9, A6-U15, T7-A14 (two), C9-A12). a

LNA nucleotides are shown in bold.

mation and because LNA nucleotides, in addition, steer the sugar conformations of neighboring nucleotides, predominantly in the 3′-direction, into N-type (C3′-endo) sugar conformations. Indeed, in the LNA3:RNA hybrid, all nonterminal deoxyriboses were found with N-type sugar puckers, even though deoxyriboses possess an inherent preference for S-type sugar puckers, and hence this hybrid adopts an A-like duplex geometry. We now extend the studies of this nonamer LNA:RNA series with the high-resolution structure of the fully modified hybrid. The structure was determined employing NOE-derived distance restraints in a simulated annealing scheme. With respect to the LNA:DNA hybrid, we have hitherto studied a number of LNA:DNA hybrids with varying numbers of LNA monomers incorporated in one of the strands. As for LNA:RNA hybrids, a conformational tuning of neighboring deoxyriboses is observed, albeit in a less pronounced manner, as it requires two flanking LNA monomers to shift the deoxyribose pucker into a pure N-type conformation. For LNA:DNA hybrids, we also observe a slight increase in N-type conformation of the deoxyriboses on the base-paired strand. We extend here our studies of LNA:DNA hybrids, with the characterization of a fully modified hybrid by analysis of 2D NMR spectra. In addition, we have determined the sugar puckers of the deoxyribose strand by analysis of COSYtype spectra. MATERIALS AND METHODS

Sample Preparation. The modified oligonucleotide was synthesized as described elsewhere (6, 20). The DNA oligonucleotide was purchased from DNA Technology, Århus, Denmark, and the RNA oligonucleotide was synthesized at University of Copenhagen. All oligonucleotides were purified by site-exclusion on a Sephadex G15 column. The samples were obtained by dissolving equimolar amounts of the two single strands in 0.5 mL of 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, and 0.05 mM NaEDTA. For experiments carried out in D2O, the duplex solutions were lyophilized three times from D2O and redissolved in 99.96% D2O (Cambridge Isotope Laboratories). Mixtures of 90% H2O and 10% D2O (0.5 mL) were used for experiments examining exchangeable protons. The final concentration of the hybrids were 0.8 mM (LNA:RNA) and 2.0 mM (LNA:DNA). NMR Experiments. NMR experiments were performed on either a Varian INOVA 500 spectrometer or a Varian INOVA 800 spectrometer at 25 °C. For the LNA: RNA hybrid the following spectra were obtained: NOESY spectra (mixing times: 80 and 250 ms) were acquired at 800 MHz in D2O using 1110 t1-experiments with 24 scans, 2048 complex points in t2, a pulse repetition time of 2.5 s, and spectral widths of 8000 Hz. The States phase cycling scheme was used, and the residual signal from HOD was removed by low-power presaturation. An inversion recovery experiment to extract the T1 relaxation rates was also obtained at 800 MHz. The remaining

spectra were all obtained at 500 MHz. A NOESY spectrum in H2O (mixing time: 200 ms) using the WATERGATE NOESY pulse sequence with 2048 complex points in t2, 900 t1-experiments with 112 scans, and a spectral width of 10000 Hz. TOCSY spectra (mixing times: 60 and 90 ms) using 1024 complex points in t2 and 600 t1experiments with 42 scans. A DQF-COSY spectrum using 4096 complex points in t2, 1024 t1-experiments with 64 scans, and a gradient 1H-13C HSQC spectrum using 2048 complex points in t2, 750 t1-experiments with 80 scans, and spectral widths of 5000 and 10000 Hz for the 1 H and 13C dimensions, respectively. For the LNA:DNA hybrid, the following spectra were obtained at 500 MHz: A NOESY spectrum (mixing time: 200 ms) in D2O using 640 t1-experiments with 96 scans, 2048 complex points in t2, a pulse repetition time of 2.4 s, and spectral widths of 5000 Hz. The States phase cycling scheme was used, and the residual signal from HOD was removed by low-power presaturation. A NOESY spectrum in H2O (mixing time: 200 ms) using the WATERGATE NOESY pulse sequence with 2048 complex points in t2, 512 t1-experiments with 128 scans, and a spectral width of 10000 Hz. A TOCSY spectrum (mixing time: 90 ms) using 1024 complex points in t2 and 512 t1-experiments with 64 scans. In addition, selective DQF-COSY and E-COSY spectra were acquired using a pulse sequence where the first pulse was replaced with an E-BURP type selective pulse (21) in order to enhance the digital resolution in F1. These spectra were acquired with spectral widths of 5000 Hz in F2 and 1200 Hz in F1, respectively. For the DQF-COSY spectrum, a total of 1472 t1-experiments were recorded, each with 64 scans, with 4096 complex points in t2. For the E-COSY spectrum, a total of 700 t1-experiments were recorded, each with 96 scans, with 2048 complex points in t2. The acquired data were processed at optimal conditions using FELIX (version 98.0, MSI, San Diego, CA). Particularly, the NOESY spectra recorded in the build-up of the LNA:RNA hybrid were linear predicted from 555 points to 750 points in t1 and baseline-corrected in F2 using the FLATT algorithm (22). Distance Restraints. A total of 333 distance restraints were obtained from 2D NOE cross-peak intensities using the method of Wijmenga et al. (23); in this variation of the isolated spin pair approximation, spin diffusion is accounted for in an average manner. NOESY cross-peak intensities from the 80 and 250 ms spectra were corrected for saturation effects using T1 relaxation times obtained from an inversion recovery experiment and were subsequently transformed to distance restraints by calibrating against all known distances. Upper and lower distance bounds were set to the calculated distances (15%. The distribution of NOE restraints for the LNA:RNA hybrid is shown in Figure 2. In total we had 166 intranucleotide, 139 internucleotide and 28 crossstrand restraints. The average width of the distance restraints was 1.50 Å, and the average restraint length 4.66 Å.

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Normal Watson-Crick base pairing was inferred from the NOESY spectrum acquired in H2O, and consequently 22 hydrogen bond distance restraints were included in the calculations. Target values for these restraints were taken from crystallographic data (24). All distance restraints were incorporated into the AMBER potential energy by a flat-well pseudopotential with the form:

{

ENOE ) -KNOEr + (r1 - 0.25 Å)KNOE, KNOE(r - r1)2, 0, KNOE(r - r2)2, KNOEr + (r2 + 0.25 Å)KNOE,

if r e r1 - 0.5 Å if r1 - 0.5 Å < r < r1 if r1 e r e r2 if r2 < r < r2 + 0.5 Å if r g r2 + 0.5 Å

where the force constants, KNOE, are in units of kcal/(mol Å) or kcal/(mol Å2), and r1 and r2 are the lower and upper distance bounds, respectively. Structure Calculations. All calculations were performed with the AMBER6 suite of programs (25) on SGI/ O2 workstations. A simulated annealing (SA) protocol was utilized to obtain the structure of the hybrid. The starting structure (A-form duplex) was obtained with the nucgen module of AMBER6. The appropriate nucleotides were modified to LNA nucleotides, and atomic charges for the modified nucleotides were calculated using the RESP procedure (26). A table with atomic charges for the four LNA nucleotides is included in Supporting Information. Initially the starting structure was restrained energy minimized before being subjected to 28 ps of molecular dynamics in time-steps of 1 fs: 4 ps at 600 K followed by cooling to 250 K over 24 ps. Finally, a further restrained energy minimization was carried out. In the SA scheme, the force constant, KNOE, was ramped from 50 kcal/(mol Å2) to 250 kcal/(mol Å2) over the initial 2 ps, kept constant at 250 kcal/(mol Å2) from 2 to 8 ps, decreased to 50 kcal/ (mol Å2) from 8 to 12 ps, and afterward kept constant at 50 kcal/(mol Å2). A distance dependent dielectric constant,  ) 4r, was used and the nonbonded cutoff was 30 Å. Deoxyribose Ring Conformations. The H1′ to H2′ and H2′′ region of the selective DQF-COSY and E-COSY spectra, respectively, was used as input to CHEOPS (27) to obtain the values of the J coupling constants for the H1′, H2′, H2′′, and H3′ deoxyribose ring protons. The deoxyribose ring conformations were analyzed assuming a fast two-state equilibrium between N- and S-type conformations with a modified version of the program PSEUROT (28). The standard version of PSEUROT obtains the best fit of the parameters PN, PS, Φmax,N, Φmax,S, and %N to the experimental coupling constants. PN,S and ΦN,S are the pseudorotation angle and puckering amplitude, respectively, of the N- and S-type conformation and %N is the mole fraction of N-type conformation. In our randomized version of PSEUROT, we include the effect of uncertainties in the determined set of coupling constants by generating 1000 sets of coupling constants according to a normal distribution in which the dispersion is set according to the uncertainty of each determined coupling constant. Uncertainties of the coupling constants were carefully assessed by how well the back-calculated spectra correlated with the experimental spectra and by the difference in the coupling constants obtained by analysis of the two types of COSY spectra. Subsequently, PSEUROT calculations were performed for all the generated sets of coupling constants and the mean values and

Table 1. Melting Temperatures of LNA:RNA and LNA:DNA Hybrids. Unmodified Duplexes of Identical Base Sequence Are Included for Comparisona

duplex LNA:RNA LNA3:RNA LNA1:RNA DNA:RNA RNA:RNAb

∆Tm per Tm, modification, °C °C 74 52 37 27 38

5.2 8.3 10.0 -

duplex LNA:DNA LNA3:DNA DNA:DNAb RNA:DNAb

∆Tm per Tm, modification, °C °C 62 44 28 27

3.8 5.3 -

a T is the duplex melting temperature and ∆T the increase m m in melting temperature per LNA monomer compared with that of the corresponding unmodified duplex. b In these duplexes, one of the terminal base pairs is switched, i.e. C-G to G-C, relative to the duplexes in this work. These data are from S. K. Singh and J. Wengel (1998) Chem. Commun., 1247-1248.

standard deviations of the parameters returned by PSEUROT were calculated. Thermal Stability Studies. The thermal stability of the hybrids was determined spectrophotometrically with a spectrophotometer equipped with a thermoregulated Peltier element. The hybridization mixtures were prepared by dissolving equimolar amounts (ca. 2.5 µM) of the oligonucleotides in 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, and 0.05 mM EDTA. The absorbance at 260 nm was monitored while the temperature was raised linearly from 10 to 80 °C (1 °C/min). The melting temperatures (Tm values) were obtained as the maxima of the first derivative of the melting curves. Circular Dichroism Spectra. A CD spectrum of the LNA:RNA hybrid from 350 to 200 nm was measured on a JASCO 710/720 spectropolarimeter. The sample was prepared by dissolving equimolar (65 µM) amounts of the oligonucleotides in 10 mM sodium phosphate buffer (pH 7.0) and 0.05 mM EDTA. The spectrum of the buffer solution was subtracted from the spectrum. The molar CD of the duplex was calculated from eq 1 (29, 30), where θ is the measured ellipticity in degrees, L is the path length in centimeters, and C is the concentration of the duplex. An absorbance spectrum was measured on a Lambda 17 spectrophotometer. The concentration of the duplex was determined from the absorbance at 260 nm using an extinction coefficient  ) 161.800 M-1 cm-1.

∆ )

θ 32.98CL

(1)

Protein Data Bank Accession Codes. Coordinates and restraints employed in calculations have been deposited in the Protein Data Bank (accession code: 1h0q). RESULTS

Thermal Stabilities. The thermal stabilities of the two hybrids studied were determined and compared with those of hybrids with isosequential base composition (Table 1). Sharp monophasic transitions were obtained with hyperchromicities of 1.2-1.3. No indications of biphasic transitions were detected. For the fully modified LNA:RNA and LNA:DNA hybrids, we observe increases in the melting temperatures of 5.2 °C and 3.8 °C per modification relative to the unmodified DNA:RNA and dsDNA reference duplexes, respectively. These results emphasize the substantial increases in thermal stability displayed by LNA modified oligonucleotides when hybridized with complementary RNA or DNA. Spectral Analysis. The 1D 1H NMR spectra of the two hybrids both show exclusively lines from the expected LNA:RNA and LNA:DNA hybrids at 25 °C. No signs of

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Figure 3. The aromatic to H1′ region of the NOESY spectrum with a mixing time of 250 ms of the LNA:RNA hybrid. The sequential H1′(n-1)-H6/8(n)-H1′(n) connectivity pathways are indicated; unbroken line for the LNA strand and broken line for the RNA strand. Adenine H2 resonances are shown with dotted lines.

alternative hybridization were observed. The NOESY spectra of both hybrids display the characteristic connectivities of right-handed nucleic acid duplexes with an overall A-type geometry, as gauged from cross-peaks involving adenine H2 protons, and with all the nucleobases in the anti conformation. The assignment of the nonexchangeable protons was performed using standard methods (31-34). The NOESY spectra with short mixing times allowed unambiguous assignments of the H2′ and H2′′ resonances. The H6′ and H6′′ resonances in the 2′O,4′-C-methylene bridge of the LNA nucleotides were assigned by their strong intranucleotide cross-peaks to H1′ of the modified nucleotides. The aromatic to H1′ region of the 200 ms NOESY spectrum of the LNA:RNA hybrid is shown in Figure 3. The NOESY spectra acquired in H2O exhibited normal Watson-Crick connectivities and were employed in the assignment of the exchangeable protons. Selections of chemical shift values for the hybrids are included in Supporting Information. Furanose Ring Conformations in the LNA:DNA Hybrid. All nucleotides in the LNA strand are found in N-type conformations as shown by the absence of COSY cross-peaks between the H1′ and H2′ protons due to cancelation of anti-phase lines (JH1′H2′ < ∼2 Hz). The favorable spectral dispersion of the H1′, H2′, and H2′′ resonances of the deoxyriboses allowed simulation of all cross-peaks and consequently determination of sugar puckers for the entire DNA strand. In Figure 4, a comparison between the experimental DQF-COSY spectrum and the spectrum obtained by spectral simulations with CHEOPS is shown. Very good agreement between the simulated and the experimental spectrum is observed. The simulated E-COSY spectrum of the hybrid agreed equally well with the experimental one. The coupling constants returned by CHEOPS were used as input for our randomized version of PSEUROT. The uncertainties of the coupling constants were gauged by monitoring the dependence of the simulation procedure on alterations in the input parameters and the difference in the coupling constants obtained from each of the two types of spectra. The coupling constants obtained, molar fractions of S-type sugar conformations, and pseudoro-

Figure 4. Comparison of the H1′ to H2′/H2′′ regions of the calculated (left) and the experimental (right) DQF-COSY spectra for the LNA:DNA hybrid.

tation angles calculated for the LNA:DNA hybrid are given in Table 2. We tried to fit the experimental coupling constants to a one-state model; however, the RMSD between calculated and experimental coupling constants was on average 1.9 Hz, which is more than the uncertainty on the experimental coupling constants. When fitting with a two-state model, the average RMSD was 0.5 Hz. In the two-state model, we allowed three parameters (the two pseudorotation angles and the molar fraction of N-type conformation) to be fitted, while in the one-state fit only two parameters (the pseudorotation angle and the puckering amplitude) were free. Obviously, the larger number of degrees of freedom in the two-state fit dictates a lower RMSD than in the one-state fit; however, we think that the decrease observed in RMSD is significant although we did not perform any statistical analysis. All deoxyribose sugars in the LNA:DNA hybrid possess sugar puckers in equilibria between N- and S-type conformations. The population of S-type conformation ranges from 57% (A12) to 80% (A17) with the populations

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Table 2. Coupling Constants for the Deoxyribose Sugar Protons in the LNA:DNA Hybrid and the Sugar Conformations Deriveda J1′2′/Hz J1′2′′/Hz J2′3′/Hz J2′′3′/Hz G10 C11 A12 T13 A14 T15 C16 A17 G18

6.1 5.7 5.2 6.1 5.7 6.5 7.0 8.7 8.4

4.7 6.4 6.2 6.4 6.4 6.5 6.3 5.4 6.4

4.7 4.3 4.5 5.5 4.7 4.9 4.7 4.6 5.7

3.8 4.0 4.3 4.0 4.0 3.8 3.8 3.0 2.4

%Sb 62 (7) 62 (9) 57 (8) 60 (9) 62 (9) 65 (9) 68 (8) 80 (7) 76 (13)

PS, degc PN, degd 165 (12) 185 (15) 183 (16) 172 (25) 183 (18) 178 (18) 175 (16) 164 (12) 160 (28)

-28 (16) -11 (17) -16 (17) -9 (33) -14 (21) -5 (32) -5 (26)

Table 3. Structural Parameters for the LNA:RNA Structurea structure EAMBER (kcal/mol) ENOE (kcal/mol) ∆dav (Å) RMSD (Å) A-type NMR

208.8 98.4

412.6 8.2

0.058 0.023

1.06 0.64b

a Force field (E AMBER) and restraint (ENOE) energies, average restraint violations (∆dav), and atomic RMSD for the A-type starting structure and the NMR structure. All-atomic RMSDs were calculated for all atoms of the seven internal base pairs. b Average pair-wise atomic RMSD for the 20 structures calculated.

a The coupling constants were derived from the H1′-H2′ and H1′-H2′′ cross-peaks in the selective DQF-COSY and E-COSY spectra using CHEOPS. The J2′2′′ coupling constants returned were between -14.2 and -15.0 Hz, values typical for deoxyribose. The sugar conformations were calculated using a randomized version of the PSEUROT program. In PSEUROT calculations, the puckering amplitudes were fixed at 36°. The standard deviations are given in brackets. b Percent S-type sugar conformation calculated using PSEUROT. c Pseudorotation angle calculated for the S-type sugar conformation. d Pseudorotation angle calculated for the N-type sugar conformation. If the mole fraction of the N-type conformer was calculated to be below 25%, the P value was not considered to be determined with sufficient accuracy.

Figure 6. Stereoview of the LNA:RNA structure viewed into the minor groove. For clarity, only hydrogens on the 2′-O,4′-C LNA methylene bridge are shown. The coloring scheme: nucleobases, yellow; sugar-phosphate backbone, red; 2′-O,4′-C methylene bridges, blue.

Figure 5. The CD spectra of various DNA:RNA and LNA:RNA hybrids of identical base sequence; DNA:RNA (dotted line), LNA1:RNA (thin line), LNA3:RNA (dashed line), and the fully modified hybrid (thick line).

being fairly uniform (at 60-65%) except for the two 3′terminal nucleotides, A17 and G18, which possess slightly higher populations of S-type puckers than the remaining nucleotides in the DNA strand. This is also shown by the slightly larger JH1′H2′ coupling constants for these nucleotides (see Table 2). CD Spectra. The CD spectrum of the fully modified LNA:RNA hybrid is shown in Figure 5 along with the spectra of the unmodified reference hybrid and the LNA1: RNA and LNA3:RNA hybrids. All of the hybrids adopt an overall A-type duplex geometry as evidenced by the negative bands at ∼210 nm and the positive bands at ∼260 nm. These bands become progressively more intense upon introduction of either one or three LNA modifications in accordance with our previous structural studies showing an increase in the A-like character of these duplexes (12, 19). For the fully modified LNA:RNA hybrid, the band at ∼260 nm has approximately the same intensity as for the hybrid with three modifications. This band is progressively blue-shifted with an increasing number of LNA monomers. The band at ∼210 nm is even more intense for the fully modified hybrid than for any of the other hybrids. Altogether, the CD spectrum of the fully modified LNA:RNA hybrid points to a regular A-like duplex structure.

Structure of the Fully Modified LNA:RNA Hybrid. The high-resolution structure was generated starting from a canonical A-type duplex geometry (see Materials and Methods). The use of an A-type structure as starting structure is justified as the N-type conformation of both the LNA and RNA nucleotides is demonstrated by the absence of H1′-H2′ cross-peaks in COSY spectra. A total of 20 final structures were calculated using the protocol described by randomly varying the initial atomic velocities. These structures all converged to one family of structures with an average pair-wise all-atomic RMSD of 0.85 Å (0.64 Å for nonterminal base pairs). Only one distance violation in excess of 0.2 Å was observed, and in addition the hybrid features a low distance restraint energy (see Table 3) and low distance violations as compared to the starting structure. General Description of the Structure. As suggested by the CD spectrum and by inspection of the NOESY spectra, the fully modified LNA:RNA hybrid has the overall appearance of an A-type duplex; for example, the minor groove is very wide and shallow, and the base pairs are displaced relative to the long axis of the helix. The RMSD between the calculated NMR structure and the energy-minimized A-type starting structure is 1.1 Å. The 2′-O,4′-C methylene bridges of the LNA nucleotides are located at the brim of the minor groove where they pose no steric hindrance for duplex formation (see Figure 6). All glycosidic angles are in anti conformations and Watson-Crick base pairing is maintained for all base pairs. The hybrid is regular as shown by the nearly straight helix axis as calculated by CURVES 5.2 (35, 36). Thus, the structure shows that a nucleic acid strand composed entirely of LNA monomers can be hybridized with an RNA strand and still retain a normal Watson-

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Table 4. A Selection of Helix Parameters for the LNA:RNA Hybrid. Values for Standard A- and B-Type Duplexes Are Included for Comparison X-disp (Å)

Y-disp (Å)

tip, deg

inclination, deg

prop. twist, deg

C1 T2 G3 A4 T5 A6 T7 G8 C9

-5.8 -6.0 -5.7 -5.8 -5.9 -5.9 -5.8 -5.7 -6.0

0.0 -0.1 -0.2 -0.3 -0.4 -0.4 -0.3 -0.3 -0.6

-5.7 -6.0 -4.9 -5.0 -6.0 -5.2 -6.5 -5.2 -4.6

13.0 12.2 12.9 11.8 13.5 13.4 8.8 5.6 2.1

-11.2 -10.9 -15.7 -13.7 -16.6 -18.2 -11.2 -10.2 -10.0

A-type B-type

-5.4 -0.7

0.0 0.0

0.0 0.0

19.1 -5.9

-13.7 -3.7

Scheme 2. Graphical Representations of Helix Parameters Discussed in the Text

C1-T2 T2-G3 G3-A4 A4-T5 T5-A6 A6-T7 T7-G8 G8-C9

twist, deg

roll, deg

rise (Å)

28.1 30.0 30.1 30.8 33.9 29.9 29.0 25.0

-0.1 5.6 -1.5 -3.3 10.4 -4.5 4.2 0.9

2.43 2.24 2.56 2.84 1.84 3.01 2.70 2.80

32.7 36.0

0.0 0.0

2.56 3.38

Table 5. Average Backbone Torsion Angles and Sugar Puckers (in degrees) in the Two Strands of the LNA:RNA Hybrida LNA RNA

R

β

γ

δ



ζ

χ

P

Φmax

-72 -78

179 181

59 61

66 79

-165 -161

-61 -68

-164 -162

13 15

60 39

a A complete table with torsion angles for individual nucleotides is included in Supporting Information.

Crick duplex framework. A stereoview of the structure is presented in Figure 6. Interstrand purine-purine stacking is observed between G3/A17, A6/A14, and G8/A12. This stacking arrangement stabilizes the inherently weaker pyrimidinepurine base pair steps in which little/no intrastrand stacking is taking place. An identical stacking pattern was observed for the previously studied LNA:RNA hybrids and for the unmodified hybrid as well, and is, as such, not a property specific for LNA modified hybrids but rather a sequence-specific property (19). The minor groove width of the hybrid is uniform along the helix ranging from 9.7 to 10.3 Å, with the average being 10.0 Å. Helix Parameters. The global helix axis and helical parameters were calculated with CURVES 5.2. The global helix axis is rather straight with a slight bend at the T5pA6 base pair step. In general the helix parameters are uniform along the duplex, with slight variations toward the 3′-end of the LNA strand for some parameters and some minor variations in the T5pA6 base pair step as a consequence of the slight bend in the helix axis. A selection of the helical parameters is given in Table 4, and graphic representations of the helical parameters discussed are shown in Scheme 2. X-displacement, Ydisplacement, inclination, and tip are the helix parameters with which the global helix axis is determined. X-displacement, with an average value of -5.8 Å, and inclination, with an average of 10.4°, are both close to the values expected for an A-type duplex. The Y-displacement and tip parameters both show that the duplex is regular in its geometry. Other parameters indicative of the A-type geometry are the average propeller twist of -13.1° and the average rise of 2.55 Å. Rise shows some variations in the A4pT5, T5pA6, and A6pT7 steps, which is caused by the curvature of the helix axis at this point. This decreases rise for the T5pA6 step which, in turn, is balanced by increases in the two juxtaposed base pair steps. The kink in the helix axis is shown by a roll of 10° in the T5pA6 step, and a negative value of opening for

A6 shows that the axis is bending toward the major groove. The average base pair twist is 29.6°, and this corresponds to a slight unwinding of the helix (for a canonical A-type duplex geometry, the base pair twist is 33°). In the corresponding unmodified DNA:RNA hybrid and the LNA3:RNA hybrid, we found average twists of 32.8° and 33.4°, respectively (19). As such, a slight unwinding of the helix might be a consequence of the fully modified LNA strand. Sugar Conformations. All nucleotides in the hybrid adopt N-type sugar puckers, the average pseudorotation angles being 13° and 15° for LNA and RNA strands, respectively. The corresponding puckering amplitudes are 60° and 39°, respectively. The sugar puckers are experimentally corroborated by vanishing H1′-H2′ crosspeaks (JH1′H2′ < ∼2 Hz) in DQF-COSY spectra. The very large puckering amplitude for the LNA sugars is a consequence of the locked bicyclic ring system. The large puckering amplitude shifts the δ backbone angle to 66° (see below). Backbone Geometry. The regularity of the duplex is accentuated by analysis of the backbone torsion angles as these all lie in the standard genus for an A-type duplex geometry, that is (R to ζ) gauche-, trans, gauche+, gauche+, trans, gauche-, with only very slight variations along each of the strands (see Table 5). In the LNA strand, the average δ angle is 66° (as compared to 79° in the RNA strand) owing to the large puckering amplitude of the LNA furanoses (see above). This alteration of 13° is absorbed by slight changes in the  and ζ angles and appears not to translate into any global perturbation of the duplex geometry. For the ribose nucleotides C11, A12, and U13, we observe that in some of the 20 structures calculated for the hybrid, the R and γ torsion angles adopt R,γ:trans,trans conformations. This is a consequence of the dynamic nature of the nucleic acid sugar-phosphate backbone and because both the R,γ:gauche-,gauche+ and R,γ: trans,trans backbone conformations are allowed by the NOE restraints employed in the structure calculations. The glycosidic angles, χ, are found in anti conformations for all nucleotides as expected from the NOESY spectra. Again there are only minute variations along the duplex, and between the strands, with the average value being -163°.

Locked Nucleic Acid Hybrids DISCUSSION

The LNA:DNA Hybrid. The LNA:DNA hybrid exhibits NMR spectral features typical of an A-like righthanded nucleic acid duplex with normal Watson-Crick base pairs and all nucleobases in anti conformations. The chemical shifts of the hybrid are somewhat altered in the LNA strand relative to the corresponding hybrid with three LNA nucleotides incorporated (37). This is a consequence of changes in the geometry owing to the added LNA nucleotides. As for the DNA strand, the chemical shift values are fairly unchanged, which shows that the overall geometry of the strand is unaltered even though the sugar equilibria of the deoxyriboses are changed (see below). Our analysis of the deoxyribose conformations in the LNA:DNA hybrid show that they all exist in equilibria between N- and S-type puckers, with the population of S-type ranging from 57 to 80% (Table 2). In the corresponding unmodified dsDNA duplex, all deoxyriboses possessed in excess of 80% S-type conformation and in a partly modified isosequential LNA:DNA hybrid (LNA monomers at positions 2, 5 and 7), the deoxyriboses of the DNA strand possessed between 71 and 93% S-type conformation (38). Obviously, the inclusion of LNA monomers in one of the strands gradually tunes the base paired strand into larger populations of N-type sugar puckers. If analyzed with a two-state model, deoxyriboses in RNA:DNA hybrids usually possess approximately 6070% S-type sugar conformation (19, 39-42). As such, the pucker pattern of the deoxyriboses in the LNA:DNA hybrid resembles that of an RNA:DNA hybrid. Although we have not determined the structure of the fully modified LNA:DNA hybrid, it appears reasonable from the evidence presented above to assume that such a hybrid structurally resembles an RNA:DNA hybrid. That is, repuckering deoxyribose sugars, a geometry intermediate of A- and B-type (albeit closer to A-type than B-type), with, for example, a minor groove width intermediate of that of A- and B-type geometries (∼8-9 Å). There is some debate as to whether the deoxyriboses in RNA:DNA hybrids are best described by an O4′-endo sugar pucker, (43, 44) a two-state model (39, 41, 45), or a model including more than two states (46). We have in this and previous work (19) experienced that J coupling constants can generally be fitted better with a two-state N/S model than with a single-state O4′-endo model. However, a two-state N/S model does not exclude that intermediate conformations are transiently occupied. If one assumes an S-type population of 67%, the energy difference between the two low-energy conformations would be ∼0.4 kcal/mol (based on a Boltzmann distribution), which is in good agreement with gas-phase ab initio calculations on a deoxyribose model system (47). In these calculations, the O4′-endo energy barrier to pseudorotation is ∼1.5 kcal/mol. Doing a statistical weighting with these energies, Brameld and Goddard calculates that the population of the “eastern” quadrant of the pseudorotation circle is 18%. This is actually in very good agreement with the 20% O4′-endo conformation proposed by Damha and co-workers based on NOE distances in a study of an RNA:DNA hybrid (46). Summarizing, we propose that it is apt to apply a two-state model to describe the sugar pucker of deoxyriboses in RNA:DNA hybrids; however, one should interpret the two sugar puckers determined as the two low-energy conformations and keep in mind that a continuum of conformations will be populated according to a Boltzmann distribution.

Bioconjugate Chem., Vol. 15, No. 3, 2004 455

The LNA:RNA Hybrid. The chemical shifts of this hybrid show only the expected changes owing to the LNA nucleotides relative to the unmodified, LNA1:RNA and LNA3:RNA hybrids. In particular the chemical shifts of the RNA strand are almost unaltered between the four hybrids, and this shows that the conformation of the RNA strand is rather unperturbed by the increasing number of LNA nucleotides incorporated in the cognate strand. The structure of the fully modified LNA:RNA hybrid has the general appearance of an A-type duplex as indicated by the large displacement of base pairs relative to the helix axis, by a number of helical parameters, and by sugar conformations in the N-range of the pseudorotation cycle. In addition, we find a minor groove width of 10.0 Å which is typical for an A-type duplex. This is in perfect agreement with the CD spectrum of the hybrid, which displayed all the characteristics of an A-type duplex geometry. Analysis of helical parameters and backbone torsion angles show that the duplex is regular; particularly the distributions of the backbone torsion angles are very uniform along the duplex with only very small variations between the LNA and RNA strands. The slight alteration of the δ angle, as imposed by the very large puckering amplitude of the LNA nucleotides, is absorbed by the neighboring torsion angles. Altogether, we have shown that a fully modified LNA strand can be hybridized with its complementary RNA strand in the usual Watson-Crick manner; moreover, the resulting hybrid adopts an almost canonical A-type duplex geometry. Thus, LNA acts as an RNA mimic, and LNA fits seamlessly into an A-type Watson-Crick duplex framework. Fully modified LNA:RNA hybrids do not elicit RNase H cleavage of the RNA strand (48). The predominant hypothesis is that RNase H recognizes a minor groove width intermediate of A- and B-type duplexes (∼8-9 Å), and the A-like structure, with a broad minor groove, of the fully modified LNA:RNA hybrid explains the RNase H inamenability of these hybrids. We have determined the structure of the corresponding partly modified LNA:RNA hybrid with three LNA modifications (at positions 2, 5, and 7; LNA3:RNA) (19). This hybrid also adopts an almost canonical A-type duplex geometry. As such, the structural alteration introduced by the increased number of LNA monomers is small, yet the increase in helical thermostability is substantial (∆Tm ) 22 °C; although the inclusion of the two 5-methylcytosines may account for approximately 2 °C). The NMR structure does not give any clues as to the origin of this thermostability. It is very likely that the 2′-oxygen of the LNA nucleotides partake in the solvation of the minor groove. Water bridges between the O2′ of the LNA bridge and O4′ of the 3′-flanking sugar are possible, thus enhancing the minor groove hydration. Such a hydration pattern would be less pronounced in the hybrid with just three LNA nucleotides. In addition, the T1 relaxation times measured for the fully modified LNA:RNA hybrid are very long (data not shown). For example, H1′ and aromatic protons have T1 relaxation times of ∼7 s and the adenine H2 proton a T1 relaxation time of ∼8 s. T1 relaxation times are influenced by a number of experimental parameters and thus can be difficult to compare between different samples. However, the values observed for the fully modified hybrid are distinctly longer than those for the LNA3:RNA and unmodified hybrids. This, qualitatively, points to a very rigid molecule with little internal motion. This would be favorable for establishing a well ordered shell of hydration.

456 Bioconjugate Chem., Vol. 15, No. 3, 2004 CONCLUSION

We have studied two fully modified LNA hybrids, an LNA:RNA, and an LNA:DNA hybrid. In both contexts, the LNA nucleotides appear as RNA mimics. As such, LNA:RNA or dsLNA duplexes might be employed as aptamers for proteins recognizing dsRNA duplexes, and LNA:DNA hybrids as RNA:DNA aptamers, for example to probe the importance of the 2′-OH of RNA in enzymatic reactions. In addition, with their high affinity toward RNA, LNAs might be utilized to target highly structured regions of tertiary RNA structure. ACKNOWLEDGMENT

We thank The Instrument Centre for NMR Spectroscopy of Biological Macromolecules at The Carlsberg Laboratory, Copenhagen, granted by The Danish Natural Science Research Council for providing spectrometer time at the 800 MHz spectrometer. The Danish National Research Foundation is thanked for financial support and Britta M. Dahl (Department of Chemistry, University of Copenhagen) is thanked for oligonucleotide synthesis. The Nucleic Acid Center is funded by the Danish National Research Foundation for studies on the chemical biology of nucleic acids. Supporting Information Available: Tables giving RESP atomic charges for the four different LNA nucleotides, chemical shifts for the two hybrids, and minor groove widths, and a comprehensive table giving backbone torsion angles. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Freier, S. M., and Altmann, K.-H. (1997). The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically modified DNA:RNA duplexes. Nucleic Acids Res. 25, 4429-4443. (2) Manoharan, M. (1999). 2′-Carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489, 117-130. (3) Kawasaki, A. M., Casper, M. D., Freier, S. M., Lesnik, E. A., Zounes, M. C., Cummins, L. L., Gonzalez, C., and Cook, P. D. (1993). Uniformly modified 2′-deoxy-2′-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. J. Med. Chem. 36, 831-841. (4) Gryaznov, S. M. (1999). Oligonucleotide N3′-P5′ phosphoramidates as potential therapeutic agents. Biochim. Biophys. Acta 1489, 131-140. (5) Hendrix, C., Rosemeyer, H., De Bouvere, B., Van Aerschot, A., Seela, F., and Herdewijn, P. (1997). 1′,5′-Anhydrohexitol oligonucleotides: hybridisation and strand displacement with oligoribonucleotides, interaction with RNase H and HIV reverse transcriptase. Chem. Eur. J. 3, 1513-1520. (6) 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. (7) Obika, S., Nanbu, D., Hari, Y., Andoh, J., Morio, K., Doi, T., and Imanishi, T. (1998). Stability and structural features of the duplexes containing nucleoside analogues with a fixed N-type conformation, 2′-O, 4′-C-methyleneribonucleosides. Tetrahedron Lett. 39, 5401-5404. (8) Wengel, J. (1999). Synthesis of 3′-C- and 4′-C-branched oligonucleotides and the development of locked nucleic acid (LNA). Acc. Chem. Res. 32, 301-310.

Nielsen et al. (9) Petersen, M., and Wengel, J. (2003). LNA: a versatile tool for therapeutics and genomics. Trends Biotechnol. 21, 7481. (10) Kværnø, L., Kumar, R., Dahl, B. M., Olsen, C. E., and Wengel, J. (2000). Synthesis of abasic locked nucleic acid and two seco-LNA derivatives and evaluation of their hybridization properties compared with their more flexible DNA counterparts. J. Org. Chem. 65, 5167-5176. (11) Braasch, D. A., and Corey, D. R. (2001). Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA. Chem. Biol. 8, 1-7. (12) Bondensgaard, K., Petersen, M., Singh, S. K., Rajwanshi, V. K., Wengel, J., and Jacobsen, J. P. (2000). Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity. Chem. Eur. J. 6, 26872695. (13) Kurreck, J., Wyszko, E., Gillen, C., and Erdmann, V. A. (2002). Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 30, 1911-1918. (14) Koshkin, A. A., Nielsen, P., Meldgaard, M., Rajwanshi, V. K., Singh, S. K., and Wengel, J. (1998). LNA (locked nucleic acid): an RNA mimic forming exceedingly stable LNA:LNA duplexes. J. Am. Chem. Soc. 120, 13252-13253. (15) Crinelli, R., Bianchi, M., Gentilini, L., and Magnani, M. (2002). Design and characterization of decoy oligonucleotides containing locked nucleic acids. Nucleic Acids Res. 30, 24352443. (16) Vester, B., Lundberg, L. B., Sørensen, M. D., Babu, B. R., Douthwaite, S., and Wengel, J. (2002). LNAzymes: incorporation of LNA-type monomers into DNAzymes markedly increases RNA cleavage. J. Am. Chem. Soc 124, 1368213683. (17) Ørum, H., Jakobsen, M. H., Koch, T., Vuust, J., and Borre, M. (1999). Detection of the factor V Leiden mutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleic acids. Clin. Chem. 45, 1898-1905. (18) Simeonov, A., and Nikiforov, T. T. (2002). Single nucleotide polymorphism genotyping using short, fluorescently labeled locked nucleic acid (LNA) probes and fluorescence polarization detection. Nucleic Acids Res. 30, e91. (19) Petersen, M., Bondensgaard, K., Wengel, J., and Jacobsen, J. P. (2002). Locked nucleic acid (LNA) recognition of RNA: NMR solution structures of LNA:RNA hybrids. J. Am. Chem. Soc. 124, 5974-5982. (20) Singh, S. K., Nielsen, P., Koshkin, A. A., and Wengel, J. (1998). LNA (locked nucleic acids): synthesis and highaffinity nucleic acid recognition. Chem. Commun. 455-456. (21) Geen, H., and Freeman, R. (1991). Band-selective radiofrequency pulses. J. Magn. Reson. 93, 93-141. (22) Gu¨ntert, P., and Wu¨thrich, K. (1992). FLATT - a new procedure for high-quality baseline correction of multidimensional NMR spectra. J. Magn. Reson. 96, 403-407. (23) Wijmenga, S. S., and van Buuren, B. N. M. (1998). The use of NMR methods for conformational studies of nucleic acids. Prog. Nucl. Magn. Reson. Spectrosc. 32, 287-387. (24) Saenger, W. (1984) Principles of Nucleic Acid Structure, Springer-Verlag, New York. (25) Case, D. A., Pearlman, D. A., Caldwell, J. W., Cheatham, T. E., III., Ross, W. S., Simmerling, C. L., Darden, T. A., Merz, K. M., Stanton, R. V., Cheng, A. L., Vincent, J. J., Crowley, M., Tsui, V., Radmer, R. J., Duan, Y., Pitera, J., Massura, I., Seibel, G. L., Singh, U. C., Weiner, P., and Kollman, P. A. (2000) AMBER 6 University of California, San Francisco. (26) Bayly, C. I., Cieplak, P., Cornell, W. D., and Kollman, P. A. (1993). A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. J. Phys. Chem. 97, 10269-10280. (27) 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. (28) de Leeuw, F. A. A. M., and Altona, C. (1983). Computerassisted pseudorotation analysis of five-membered rings by means of proton spin-spin coupling constants: program PSEUROT. J. Comput. Chem. 4, 428-437.

Locked Nucleic Acid Hybrids (29) Woody, R. W. (1995). Circular dichroism. Methods Enzymol. 34-71. (30) Baase, W. A., and Johnson, W. C. (1979). Circular dichroism and DNA secondary structure. Nucleic Acids Res. 6, 797814. (31) 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. (32) 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. (33) Scheek, R. M., Russo, N., Boelens, R., Kaptein, R., and van Boom, J. H. (1983). Sequential resonance assignments in DNA 1H NMR spectra by two-dimensional NOE spectroscopy. J. Am. Chem. Soc. 105, 2914-2916. (34) Wu¨thrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley & Sons, New York. (35) Lavery, R., and Sklenar, H. (1988). The definition of generalized helicoidal parameters and of axis curvature of irregular nucleic acids. J. Biomol. Struct. Dyn. 6, 63-91. (36) Lavery, R., and Sklenar, H. (1989). Defining the structure of irregular nucleic acids: conventions and principles. J. Biomol. Struct. Dyn. 7, 655-667. (37) Jensen, G. A., Singh, S. K., Kumar, R., Wengel, J., and Jacobsen, J. P. (2001). A comparison of the solution structures of an LNA:DNA duplex and the unmodified DNA:DNA duplex. J. Chem. Soc., Perkin Trans. 2 1224-1232. (38) Petersen, M., Nielsen, C. B., Nielsen, K. E., Jensen, G. A., Bondensgaard, K., Singh, S. K., Rajwanshi, V. K., Koshkin, A. A., Dahl, B. M., Wengel, J., and Jacobsen, J. P. (2000). The conformations of locked nucleic acids (LNA). J. Mol. Recognit. 13, 44-53. (39) Gonza´les, C., Stec, W., Reynolds, M., and James, T. L. (1995). Structure and dynamics of a DNA. RNA hybrid duplex with a chiral phosphorothioate moiety: NMR and molecular dynamics with conventional and time-averaged restraints. Biochemistry 34, 4969-4982.

Bioconjugate Chem., Vol. 15, No. 3, 2004 457 (40) Bachelin, M., Hessler, G., Kurz, G., Hacia, J. G., Dervan, P. B., and Kessler, H. (1998). Structure of a stereoselective phosphorothioate DNA/RNA duplex. Nat. Struct. Biol. 5, 271-276. (41) Gyi, J. I., Lane, A. N., Conn, G. L., and Brown, T. (1998). Solution structures of DNA:RNA hybrids with purine-rich and pyrimidine-rich strands: comparison with the homologues DNA and RNA duplexes. Biochemistry 37, 73-80. (42) Aramini, J. M., and Germann, M. W. (1999). Solution structure of a DNA‚RNA hybrid containing an R-anomeric thymidine and polarity reversals: d(ATGG-3′-3′-RT-5′-5′GCTC)‚r(gagcaccau). Biochemistry 38, 15448-15458. (43) Fedoroff, O. Y., Salazar, M., and Reid, B. R. (1993). Structure of a DNA: RNA hybrid duplex. Why RNase H does not cleave pure DNA. J. Mol. Biol. 233, 509-523. (44) Fedoroff, O. Y., Ge, Y., and Reid, B. R. (1997). Solution structure of r(gaggacug):d(CAGTCCTC) hybrid: implications for the initiation of HIV-1 (+)-strand synthesis. J. Mol. Biol. 269, 225-239. (45) Cheatham, T. E., III., and Kollman, P. A. (1997). Molecular dynamics simulations highlight the structural differences among DNA:DNA, RNA:RNA, and DNA:RNA hybrid duplexes. J. Am. Chem. Soc. 119, 4805-4825. (46) Denisov, A. Y., Noronha, A. M., Wilds, C. J., Trempe, J.F., Pon, R. T., Gehring, K., and Damha, M. J. (2001). Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluor-ANA/RNA and DNA/RNA hybrids. Nucleic Acids Res. 29, 4284-4293. (47) Brameld, K. A., and Goddard, W. A., III. (1999). Ab initio quantum mechanical study of the structure and energies for the pseudorotation of 5′-dehydroxy analogues of 2′-deoxyribose and ribose sugars. J. Am. Chem. Soc. 121, 985-993. (48) Sørensen, M. D., Kværnø, L., Bryld, T., Håkansson, A. E., Verbeure, B., Gaubert, G., Herdewijn, P., and Wengel, J. (2002). R-L-ribo-Configured locked nucleic acid (R-L-LNA): synthesis and properties. J. Am. Chem. Soc. 124, 2164-2176.

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