Atomistic Investigation of the Effect of Incremental Modification of

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Atomistic Investigation of the Effect of Incremental Modification of Deoxyribose Sugars by Locked Nucleic Acid (β‑D‑LNA and α‑L‑LNA) Moieties on the Structures and Thermodynamics of DNA−RNA Hybrid Duplexes Gorle Suresh and U. Deva Priyakumar* Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500 032, India S Supporting Information *

ABSTRACT: Chemically modified oligonucleotides offer many possibilities in utilizing their special features for a vast number of applications in nucleic acid based therapies and synthetic molecular biology. Locked nucleic acid analogues (α-/β-LNA) are modifications having an extra ring of 2′-O,4′-C-methylene group in the furanose sugar. LNA strands have been shown to exhibit high binding affinity toward RNA and DNA strands, and the resultant duplexes show significantly high melting temperatures. In the present study, molecular dynamics (MD) simulations were performed on DNA−RNA hybrid duplexes by systematically modifying their deoxyribose sugars with locked nucleic acid analogues. Several geometrical and energetic analyses were performed using principal component (PCA) analysis and binding free energy methods to understand the consequence of incorporated isomeric LNA modifications on the structure, dynamics, and stability of DNA− RNA hybrid duplex. The β-modification systematically changes the conformation of the DNA−RNA hybrid duplex whereas drastic changes are observed for α-modification. The fully modified duplexes have distinct properties compared to partial and unmodified duplexes, and the partly modified duplexes have properties intermediate to full strand and unmodified duplexes. The distribution of BI versus BII populations suggests that backbone rearrangement is minimal for β-LNA modification in order to accommodate it in duplexes whereas extensive backbone rearrangement is necessary in order to incorporate α-LNA modification which subsequently alters the energetic and structural properties of the duplexes. The simulation results also suggest that the alteration of DNA−RNA hybrid properties depends on the position of modification and the gap between the modifications.

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toward North-form (N-form is characterized by C3′-endo and C2′-exo conformation of the sugar moiety, which is often found in A-type nucleic acids) similar to RNA duplexes.5,6 Besides high binding affinity, the single-stranded antisense oligonucleotides acting on the mRNA should also exhibit high biostability and efficient cellular uptake.7−9 Previous studies have shown that the above-mentioned features are attainable up to some extent by introducing chemical modifications at sugar and phosphate moieties. Some of the chemically modified AOs include 2′-O-methyl,10 locked nucleic acids (LNA),11−13 peptide nucleic acids (PNA),14 tricyclo-DNA,15 morpholinoNAs,16 hexitol nucleic acids (HNA),17 arabino nucleic acids (ANA), and 2′-F-arabino nucleic acids (2′-F-ANA).18 Locked nucleic acid (LNA) duplexes are chemically modified nucleic acids in which an extra ring is introduced in sugar connecting to C2′ and C4′ atoms with −O−CH2− group

INTRODUCTION Being central to transmission, expression, and conservation of genetic information, nucleic acids are perceived as effective drug targets for the treatment of many diseases.1−4 The drug therapies which target the nucleic acids (genetic level) attribute high generality and rationale than traditional drug therapies. One alternative approach is antisense therapy, where the introduced synthetic oligonucleotide (AO) binds to the mRNA with high affinity and stops the transmission of the genetic information by either forming stable duplex or degrading the target mRNA with the help of RNase H enzyme (this process is known as RNase H activity).1−4 The basic principle behind the antisense therapy is observed in nature during the gene regulation process.1,2 Since the hybrid duplexes (formed by pure DNA and RNA strands) exhibit low stability and moderate affinity toward RNase H activity, there is an immense interest in studying the chemically modified nucleic acid strands (AOs) to possibly increase binding affinity with complementary nucleic acid strands. Recent studies have been focusing on modifications which can drive the furanose sugar conformation © 2014 American Chemical Society

Received: February 11, 2014 Revised: May 5, 2014 Published: May 21, 2014 5853

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systematic incorporation of β-LNA modifications in DNA strand turns the duplex conformation toward N-type.37 Similarly, NMR studies performed on 9-mer and 10-mer DNA duplexes showed that the conformation is largely retained after introducing up to three to four α-LNA modifications.38 Structural studies had been performed on DNA−RNA hybrid duplex by introducing three α-LNA modifications in the DNA strand and observed an intermediate A/B-like conformation similar to native hybrid, suggesting a similar behavior of deoxyribose sugar to α-LNA.20 Hence, the α-LNA and β-LNA modifications were proposed as DNA and RNA mimics, respectively, in the context of helical structure. However, a recent study showed that the structure of fully modified β-LNA duplex differs significantly compared to pure RNA duplex.39 The full β-LNA modification results in decreasing twist and roll and narrowing minor groove compared to pure RNA duplex. Classical molecular dynamics (MD) simulations have been shown to be valuable tools to study nucleic acid properties.40−46 Pande and co-workers have done computational study on the fully modified β-LNA:β-LNA duplexes and explained several aspects like hydration, conformation, and internal dynamics in comparison to regular A-RNA duplexes.40 The structural differences between full β-LNA and RNA duplexes were also observed. The observed geometric differences indicate a nonregular A-type geometry for full β-LNA duplex. The recent MD simulations study on full α-LNA and β-LNA duplexes suggest that the full β-LNA duplex has a conformation similar to the regular RNA duplex but the full α-LNA duplex exhibits a conformation which is distinct from typical A- and Btype conformations despite having similar thermodynamic stability.41 Despite having several studies on the LNA-modified nucleic acid duplexes, the variations in structure, dynamics, and stability of nucleic acid duplexes upon introducing α-/β-LNA modifications are still not well understood. For example, the NMR study on a DNA−RNA hybrid duplex suggests that when three α-LNA modifications are introduced, a structure similar to a DNA−RNA hybrid with a higher melting temperature results.20 To gain a more detailed insight into the structures, dynamics, flexibility, and stability of DNA−RNA hybrids upon introducing LNA analogues (α-LNA and β-LNA) into its DNA strand, MD simulations have been performed on DNA−RNA hybrid duplex by systematically modifying its deoxyribose sugars (Figure 1). This kind of study is indispensable to understand how the chemical modifications affect the nature of the nucleic acid duplexes.

(Figure 1). Locked nucleic acid analogues such as 2′-O,4′-Cmethylene-β-D-ribofuranosyl (β-D-LNA) and 2′-O,4′-C-meth-

Figure 1. (a) Chemical representation of α-LNA and β-LNA modifications. (b) Numbering of the DNA and RNA strands of DNA−RNA hybrid used during the simulations and various analyses. (c) Sequences used to model the α/β-LNA-modified DNA−RNA hybrid duplexes studied in the present study. The modified site is indicated in orange color.

ylene-α-L-ribofuranosyl (α-L-LNA) were shown to increase the thermodynamic stability of the DNA, RNA, and DNA−RNA hybrid duplexes when they are incorporated into strands and increase the melting temperatures up to ∼10 °C per modification.19,20 The high binding affinity of LNA analogues toward other nucleic acid strands make them useful in many nucleic acid research fields covering broad range of applications in therapeutics, biotechnology and molecular biology.19,21 LNAs are utilized in DNAzymes,22 siRNA approaches,23 and molecular beacons24 and in RNA in situ hybridization.25,26 LNA-modified oligonucleotides are shown to be capable of improving inhibitor efficiency for HIV-I27 and nucleotide cleavage for hammerhead ribozymes.28 β-LNA modification and its stereoisomer α-LNA had caught much attention compared to other LNA modifications.21−23,27,30 These two isomers (α-LNA and β-LNA) differ in the orientation of the −O−CH2− group (Figure 1) but share many features: both the modifications in nucleic acid strands result in strong binding to complementary strands, show high potency as antisense oligonucleotides as found in both in vitro and in vivo experiments, and have commercial availability and ease of synthesis by use of conventional phosphoroamidite chemistry. Given the wide range of applications, it is important to understand the structure, dynamics, and stability of LNAmodified duplexes with respect to pure nucleic acids. Several attempts have been made to synthesize and characterize LNAmodified nucleic acids. NMR studies performed on βLNA:RNA and β-LNA:DNA duplexes had suggested that these duplexes display helical features similar to native nucleic acids (like Watson−Crick base pairing, base stacking, helical twist, anti orientation etc.).31,32 The NMR studies performed on 9-mer DNA−RNA duplexes containing one, two, and three LNA modifications showed increasing A-type character with respect to the number of modifications.33−36 Previous 2D NMR study showed that the change in the conformation of the backbone is local especially if the number of β-LNA modifications is less, whereas the global structure resembles DNA−RNA hybrid. Furthermore, MD simulation studies by Ivanovo et al. on LNA−DNA hybrid have shown that the

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COMPUTATIONAL METHODS Model Systems. To investigate the variations in the structure and dynamics of the DNA−RNA hybrid duplex, the sugars corresponding to one, two, three, and full strand deoxyribose nucleotides were modified with α-/β-LNA moieties (see Figure 1). Three different systems were considered for mono (LNA11, LNA12, LNA13) and di (LNA21, LNA22, LNA23) modifications according to the position and the number of nucleotides between them, respectively. The modifications were placed on the sugars of thymine present close to the 5′ terminal (LNA11), at the center (LNA12), and close to the 3′ terminal (LNA13) of the DNA strand in single-modified duplexes. The double-modified duplexes LNA21, LNA23, and LNA22 have one, two, and three nucleotide separation between two LNA modifications, respectively. The model systems were conceived in such a way

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tions were extended up to 20 ns in NPT ensemble by applying a weak harmonic restraint using a force constant 2 kcal/mol/Å2 on the hydrogen bonds of the terminal base pairs in order to prevent their chances of opening.40,41 The Leapfrog integrator was used to integrate Newton’s equations of motion that used a 2 fs integration time step. The coordinates were saved for every 5 ps for further analysis. The translational and rotational motions of the solute were removed by aligning all the frames of solute on its initial conformation. Final 10 ns simulation trajectories were used for all the structural and energetic analysis treating the first 10 ns simulation time as equilibration period based on the calculated root-mean-square deviations for duplexes with respect to the average structure obtained from final 10 ns (Figure S1 in the Supporting Information). VMD60 and Grace61 were used for image generation and making plots, respectively. The analysis corresponding to helical, base step parameters and groove widths were done with the help of Curves+ program.62

to understand the change in duplex properties with respect to the number of modifications, the position of modification and the gap between two modifications, which resulted in 16 model systems. The available DNA−RNA hybrid NMR experimental structure (1HG9)29 was used as template to generate the model systems by introducing appropriate modifications corresponding to α-/β-LNA nucleotides using CHARMM biomolecular simulation program.47 Available NMR structures were used as starting conformations for some of the LNA incorporated systems wherever available (Table 1).20,29,31,32 Control simulation on the DNA−RNA hybrid NMR experimental structure was also run using the same simulation protocol. Table 1. Protein Data Bank Ids of the Available α-/β-LNAModified DNA−RNA Hybrid Duplexes name

α-LNA

β-LNA

hybrid LNA11 LNA12 LNA13 LNA21 LNA22 LNA23 LNA3 FLNA

1HG929 − − − − − − 1OKF20 −

1HG929 − 1HHW31 − − − − 1HHX31 1H0Q32

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RESULTS AND DISCUSSION A comprehensive study has been done by performing several structural and energetic calculations to understand how the incorporated isomeric LNA modifications affect the DNA− RNA hybrid duplex properties. This section is mainly divided into four subsections: first subsection discusses the structural variations in hybrid duplex upon the incorporation of one to many LNA modifications in terms of duplex conformation, thermodynamic stability, deformation, and global conformation. The second subsection discusses the similarities/differences in α-LNA:RNA and β-LNA:RNA duplexes in comparison to the pure DNA−RNA hybrid. The final two subsections cover how different the structures are when isomeric LNA modifications are introduced at different positions and with respect to the gap between two LNA modifications in DNA− RNA hybrid duplex. The present MD results have been compared with the available experimental results whenever possible. Structural Deviations and Dynamics. Root mean square (rms) deviation values calculated for all the duplexes studied here with respect to the average structure obtained from the final 10 ns simulations and averaged for the same simulation time are presented in Table 2. The rmsd values have been calculated by considering only the non-hydrogen atoms; the extent of deviation was also calculated for the atoms of the backbone and nucleobases individually. All the structures show deviation values within the range of 0.89−2.14 Å, indicating closeness to their initial conformation. The α-LNA-modified duplexes show high deviation values than β-LNA-modified, suggesting significant difference in the duplexes compared to regular DNA−RNA hybrid. The evolutions of rms deviations with simulation time are given in Figure S1a,b in the Supporting Information. Rms fluctuation (rmsf) calculations were done to understand the change in the DNA−RNA hybrid duplex flexibility with the incorporation of α-/β-LNA modifications. The rmsf values in Table S1 in the Supporting Information indicate that the α-LNA-modified duplexes show different flexibility pattern compared to the β-LNA-modified duplexes. The α-LNA-modified duplexes show significantly high rmsf values than β-LNA-modified duplexes. The observed changes are mainly due to the high fluctuations of the DNA− RNA hybrid duplex backbone. Noticeably, α-LNA modification increases the duplex flexibility whereas the β-LNA modification decreases the flexibility of the DNA−RNA hybrid duplex,

MD Simulation Protocol. All the MD simulations were performed using the NAMD48 program employing the CHARMM36 all-atom nucleic acid force field.49−51 The topology and parameters for LNA modification were taken from the study of Pande and co-workers,40 developed by considering the point charges based on the RESP charges taken from the previous study of Nielsen et al.32 Previous studies that used these parameters on fully modified LNA duplexes obtained good agreement with the experimental studies.40,41 Missing hydrogen atoms were added to nucleic acids by using HBUILD command in CHARMM program, followed by 500step steepest decent minimization (SD) by applying massweighted harmonic constraints on heavy atoms. The minimized structures were then immersed in a pre-equilibrated water box with edge lengths 48 Å × 38 Å × 38 Å, and sodium ions were added randomly for neutralization. All water molecules making nonphysical contacts with the solute atoms were deleted based on a distance cutoff of 2 Å. A modified TIP3P52 model was used for the explicit solvent environment. These systems were subjected to a 500-step SD minimization followed by a 500-step adopted basis Newton−Raphson (ABNR) minimization. All the non-hydrogen atoms of the nucleic acid were restrained using harmonic potential with a force constant of 5 kcal/mol/ Å2. These systems were equilibrated for 100 ps under the same harmonic restraints on the nucleic acids in NVT ensemble. The covalent bonds involving hydrogens were constrained by applying SHAKE algorithm53 in all the simulations and calculations. CRYSTAL module54 was employed for setting up periodic boundary conditions. Temperature of the systems was maintained at 300 K using the Nose−Hoover thermostat,55 and constant pressure was maintained using the Langevin piston algorithm.56 The electrostatic real space and the Lennard-Jones (LJ) interactions were truncated at 12 Å with a LJ force switch smoothing function from 10 to12 Å.57 Particle mesh Ewald (PME) summation58,59 method was used to treat long-range electrostatic interactions. The production simula5855

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The hydrogen bond distances corresponding to all the base pairs present in all the duplexes were calculated. The probability distributions corresponding to the distances of central hydrogen bond (N1−N3) (Figure S2a,b in the Supporting Information) show a sharp peak at 3 Å, indicating stability of the base pairs with the maintenance of Watson− Crick (WC) base pairing. But partial base pair opening is observed for certain base pairs in some duplexes indicated by the peaks corresponding to distances longer than 4.5 Å. Backbone and Sugar Conformations. The sugar puckering angles, characteristic backbone dihedral angles α, β, γ, δ, ε, ζ, and sugar−base dihedral angle (χ) are capable of differentiating the nucleic acid conformations. To understand the backbone conformation and dynamics in modified duplexes, an additional parameter ε − ζ is reported which is generally used for characterizing BI and BII conformations (Figure 2). The backbone conformation of the DNA is BI when ε − ζ < 0, and BII for ε − ζ > 0.63,64 The previous MD simulations on full LNA-modified duplexes suggest that the α-LNA-modified duplex has backbone conformation that is different from the typical DNA, RNA, and β-LNA-modified duplex.41 The probabilities corresponding to ε − ζ were computed by using the ε and ζ dihedral angles of all the nucleotides present in the duplex (Figure 2). The probability distributions in Figure 2 indicate a systematic increase in probability in the sampling region around 60−120° (BII) in α-LNA-modified duplexes whereas such kind of increase is not seen in β-LNA-modified duplexes. This suggests that the incorporation of β-LNA modification does not require much rearrangement in backbone in order to accommodate its sugar moiety in the context

Table 2. Average Values of Root Mean Square Deviations for Non-hydrogen Atoms of Overall, Backbone (Sugar and Phosphate) and Bases (Nucleobases) Corresponding to All the Duplexes Studied with Respect to the Averaged Structure Obtained from the Final 10 nsa duplex hybrid LNA11 LNA12 LNA13 LNA21 LNA22 LNA23 LNA3 FLNA a

overall 1.15 1.22 1.51 1.16 1.85 1.14 1.35 1.20 1.45 1.24 1.43 1.11 1.40 1.29 1.43 1.34 1.73

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.05 0.04 0.02 0.18 0.02 0.08 0.04 0.06 0.02 0.19 0.06 0.09 0.04 0.03 0.01 0.11

backbone 1.26 1.33 1.69 1.29 2.14 1.27 1.50 1.35 1.71 1.38 1.51 1.24 1.48 1.45 1.55 1.44 1.84

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.06 0.06 0.03 0.23 0.02 0.07 0.05 0.08 0.03 0.14 0.07 0.10 0.04 0.04 0.02 0.11

bases 0.95 1.02 1.07 0.92 1.28 0.89 1.06 0.93 1.00 0.97 1.21 1.38 1.19 1.01 1.20 1.11 1.45

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.01 0.03 0.02 0.01 0.08 0.01 0.08 0.03 0.02 0.02 0.26 0.03 0.07 0.03 0.02 0.01 0.10

Normal letters, β-LNA, italics, α-LNA).

agreeing with the previous MD simulation results on β-LNAmodified duplexes.40 Some additional discussions on the stiffness/flexibility of the DNA−RNA hybrid duplex will be discussed in the later sections.

Figure 2. Probability distributions of dihedral angles (ε − ζ) for all the α-LNA (left) and β-LNA (right) modified DNA−RNA hybrid duplexes. The difference (ε − ζ) corresponding to pure DNA−RNA hybrid duplex (black) and observed in experimental structures (green) is also included in all the sections for reference. 5856

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Figure 3. (a) Pictorial representation of pseudorotation angles sampled by α-/β-LNA-modified, DNA, and RNA duplexes. (b) Percentage of 2E + 3E (green) and 3E (blue) conformations of DNA strand of the DNA−RNA hybrid duplex when α-/β-LNA modifications are incorporated. The 2E + 3E (black) and 3E (red) conformations of pure DNA−RNA hybrid duplex are also shown in both the panels for comparison.

slight transformation from high anti to anti regions as the number of LNA modifications (for both isomers) increases. There were no appreciable changes observed in backbone dihedral angles as the increase in number of β-LNA modifications whereas the backbone sample additional regions when incorporating α-LNA modifications and their probability increases with the increase in number of modifications. Generally, ribose sugars exhibit 3E conformation while deoxyribose sugar exhibits 2E conformation in addition to minor sampling in 3E and 0E conformations (Figure 3). Previous studies have shown that the ribose sugars present in DNA−RNA hybrid duplex exhibit conformations similar to the conformations exhibited by the ribose sugars of pure RNA duplex but a dynamic equilibrium exists between 2E and 3E

of WC base pairing and better stacking. However, the incorporated α-LNA moiety rearranges the nucleic acid backbone in order to adjust its sugar moiety into the WC pairing. This rearrangement sometimes causes destabilization in the DNA−RNA hybrid duplex through opening of the same and neighbor base pairs. Full strand modified duplexes are stable for both the modifications irrespective of the change in backbone conformation. To further understand the impact of incorporated isomeric LNA analogues on the DNA−RNA hybrid duplex conformation, the probability distributions corresponding to all the backbone dihedral angles are presented in Figure S3a,b in the Supporting Information. The distributions of glycosidic bond dihedral angle (χ angle) indicate that all the bases are in anti conformation despite a 5857

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of relation is not observed for α-LNA-modified duplexes. Some of the α-LNA-modified duplexes (LNA11 and LNA12) exhibit wider minor groove width values than pure DNA−RNA hybrid duplex. The internal dynamics of the nucleic acid duplexes depends on the stacking of nitrogenous bases present in the same strand and complementary strand and these are crucial for the duplex stability. This base stacking can be explained by using helicoidal and step parameters. Helicoidal parameters represent the orientation of base pair relative to its global helical axis, and step parameters give the orientation and position of base pair with respect to next base pair. The average values of the helical parameters shown in Figures S4 and S5 in the Supporting Information indicate that the β-LNA modification changes the duplex structure in a systematic manner whereas α-LNA modification shows drastic changes in the duplex structure. All the helical parameters, especially shift, roll, and twist, are more sensitive to the structural modifications similar to the previous MD studies on modified nucleic acid duplexes.37,69 The β-LNA modification imparts a systematic variation in most of the parameters whereas drastic changes were observed for α-LNA modifications. For example, the twist of the helix decreases as the increase in number of α-LNA modifications compared to βLNA modifications. Helical unwinding in the DNA−RNA hybrid duplex is observed for partial α-LNA modifications. The duplexes with full α-/β-LNA-modified strands exhibit average values that are distinct from partially modified duplexes (Figure S5). The average values corresponding to individual base pair step parameters indicate that partial modifications corresponding to these α/β-modifications perturb the duplex locally (Figures S6 and S7 in the Supporting Information). But the effect is significantly higher for α-LNA modification resulting in change in the global structure of the DNA−RNA hybrid duplex. The variations observed in base pair step parameters indicate that the LNA modifications show different impact on the duplex internal dynamics. These dynamics are independent of the number, position, and gap between two modifications. But the duplexes with full LNA strand exhibit properties that differ from partly modified duplexes and the observed changes adequately reflect this. Some of the internal rotations (twist) and translational (shift) motions are considerably dependent on the number of modifications and support the fact that the partly modified duplexes have intermediate properties than pure and fully modified duplexes. Though significant variations are observed in some of the helical parameters of partly β-LNAmodified duplexes, the overall effect is still the same, implying that the changes due to partial β-LNA modification are local, supporting the experimental results.34−36 Change in Thermodynamic Stability. The stability of helix depends primarily on the interactions among WC base pairs and stacking interactions with respect to other bases present in the same strand and its complementary strand. Binding free energy calculations were performed using the MM-GBSA method to understand the change in thermodynamic stability of DNA−RNA hybrid when incorporating various numbers of isomeric LNA modifications. In general, the total free energy can be determined using the following equation: G = EMM + Gsolv − TSMM, where G represents total free energy, EMM represents the total molecular mechanical energy, Gsolv gives contribution from solvent, T is absolute temperature, and SMM represents the molecular mechanical entropy. The binding free energy between two complementary strands can be calculated as ΔGbinding = Ecoul(DNA−RNA) +

conformations for the deoxyribose sugars, resulting in an intermediate A/B-like conformation to the DNA−RNA hybrid duplex.45,65 Experimental studies have suggested that the α- and β-LNA analogues act as DNA and RNA mimics, respectively.20 The percentages of puckering angles corresponding to DNA strand were calculated for all the duplexes in order to understand the variation in conformation of the pure and modified DNA−RNA hybrid duplex (Figure 3). The changes observed in percentages in Figure 3 indicate that the systematic incorporation of α- and β-LNA modifications progressively turn the DNA strand toward 3E and 3E conformations, respectively. But the impact of α-LNA modification is high compared to its analogue β-LNA. Percentages corresponding to the individual sugar conformations in all the duplexes are provided in the Supporting Information (Table S2). The sugar puckering amplitudes, the maximum torsion angle with respect to other atoms in the plane, were calculated to check the differences in sugar puckering due to LNA modifications (data not shown). It is noticed that the incorporated LNA increases the sugar moiety amplitude with the increase in sugar amplitude of the complementary nucleotide. However, these two modifications act differently on the neighbor sugar moieties; i.e., the α-LNA modification increases their amplitudes and the β-LNA modification decreases it. This could be due to the orientation of the added −O−CH2− group. The observed changes in conformation and amplitudes of the sugars are due to incorporation of LNA modifications and they indicate instability of the DNA−RNA hybrid duplex in terms of the conformation. Minor Groove Widths and Helical Parameters. The previous studies have suggested that the intermediate minor groove width of DNA−RNA hybrids act as one of the key factors in the RNase H activity.66 Average values of minor groove width regions present in all the pure and modified DNA−RNA hybrid duplexes were calculated and are shown in Figure 4. These values indicate that the β-LNA modification

Figure 4. Average values of minor groove width regions for β-LNA (red) and α-LNA (black) modified duplexes calculated for the final 10 ns simulation time.

does not show appreciable change in the minor groove width whereas the α-LNA modification narrows the minor groove width. The extent of impact on the minor groove width is high for α-LNA modification than β-LNA modification. The minor groove width regions of the partial β-LNA-modified DNA− RNA hybrid duplexes are very close to the minor groove width values observed for the full β-LNA duplex41 whereas such kind 5858

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Evdw(DNA−RNA) + Gsolv(DNA−RNA) − Gsolv(DNA) − Gsolv(RNA); here the double strand represents duplex and DNA, RNA represents individual strands. The solvation free energy was estimated as the sum of the electrostatic solvation energy (GGB) and the nonpolar solvation energy (Gnonpolar). Gsolv = GGB + Gnonpolar; the electrostatic contribution to the solvation free energy was estimated by using the generalized Born molecular volume (GBMV) method. The nonpolar contribution toward solvation free energy was estimated as Gnonpolar = γ × SASA, where γ = 0.0072 kcal/mol/Å2 and SASA is the solvent-accessible surface area,67,68 calculated by using a probe particle with a radius of 1.4 Å on DNA−RNA surface. The contribution from entropy is not considered here and such approach is widely used in the literature and has been found to be adequate for obtaining qualitative trends.43,69−71 Within the approximations used here, the entropies of the individual strands are similar when they are part of the duplex and when they are free/considered individually. Hence, including the entropic factors does not change the qualitative trends (see Figure S8 in the Supporting Information). Relative binding free energies (ΔΔG) with respect to the DNA−RNA hybrid were calculated (Figure 5).

to understand the helical stiffness of the modified DNA−RNA hybrid duplexes. Diagnolization of positional covariance matrices provide eigenvectors and eigenvalues where eigenvectors describe the nature of the essential movements and eigenvalues give the contribution of each movement to the positional variance of the trajectory. The force constants calculated for essential modes (in this case, first 15 modes) represent the rigidity of the duplex. It has been shown before that the 10 lowest modes account for most of the fluctuations of nucleic acid duplexes.45,65,72,73 The force constants, within the harmonic limit, can be calculated from the eigenvalues with the following relation ki =

kBT λi

where λi represents the eigenvalue (in Å2) corresponding to mode i, kB is the Boltzmann constant, and T is the absolute temperature. The calculated force constants indicate similar plasticity of the duplexes at small deformation modes (Figure 6). But the variations in force constants become different at higher modes for α-/β-LNA-modified duplexes, indicating their different duplex nature in terms of the helix stiffness. It is also noticed that the duplexes with fully modified α-/β-LNA strand are softer than partially modified and pure DNA−RNA hybrid, agreeing with the experimental results. As the number of α-/βLNA modifications increases in the DNA−RNA hybrid, the duplex becomes softer and less stiff than the regular DNA− RNA hybrid. The duplex with three LNA modifications is less stiff than the full strand modified duplex, further supporting the experimental results and validating the MD simulations. The intramolecular entropies were calculated employing the Schlitter method by using the eigenvalues obtained from the mass-weighted covariance matrix.45 These plots indicate that the DNA−RNA hybrid duplex is the most disordered structure at smaller essential modes. But this situation changes at higher modes for both α-/β-LNA modifications (Figure 6). Even though the stiffness analysis using essential motions provides useful information regarding deformability, the modes of such motions are not easily understandable.72,74 The elastic force constants corresponding to the helix deformations along their translational and rotational motions in helical space (shift, rise, slide, roll, tilt, and twist) were calculated to examine the nature of fluctuations. The inversion of the helical covariance matrix (C = ⟨XiXj⟩) of helical parameters (Xi,Xj) gives the stiffness matrix (F = F[kij]), and the diagonal elements (kij, i = j) give force constants corresponding to pure steps and offdiagonal elements (ki,j, i ≠ j) representing the force constant for cross terms.45,72

Figure 5. Relative binding free energies (in kcal/mol) for DNA−RNA hybrids with increasing incorporated LNA modifications.

These ΔΔG values indicate that the full strand modified DNA−RNA hybrid duplexes are more stable than pure hybrid duplex, in agreement with the experimental results.32 Some of the partial β-LNA-modified duplexes are also more stable than pure DNA−RNA hybrid, agreeing with the previous studies. However, the partial incorporation of α-LNA modifications results in duplexes that are unstable compared to pure DNA− RNA hybrids. Further, it is observed that the enhanced duplex stability via incorporating α-/β-LNA modifications is dependent on the position and gap (number of nucleotides) between two successive LNA modifications. It is also noticed that the increased LNA modifications in DNA strand show strong affinity toward complementary RNA strand and favorable LNA−RNA duplex formation for β-LNA modification whereas the reverse is observed for partial α-LNA modification. Helical Stiffness/Deformability and Effect of LNA Modifications. Previous experimental studies showed that the LNA−RNA hybrid is more rigid than pure duplex and the fully modified LNA strand makes the duplex more rigid than partially modified LNA strand.18 Force constants and intramolecular entropies in harmonic limit (along essential modes) were computed by using principal component analysis (PCA)

F = kBTC −1

where F represents the stiffness matrix, kB is the Boltzmann constant, T is absolute temperature, and C represents the covariance matrix. To further explain the stiffness at the global translational (ktrans), rotational (krotat), and global (kglobal) levels, the respective force constants are calculated by using the following equations. k trans = kshiftk risekslide

k rotat = k rollk twistk tilt kglobal = k transk rotat 5859

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Figure 6. Harmonic force constants (in kcal/mol/Å2) and intramolecular entropies (in kcal/mol/K) corresponding to the first 15 modes for all the duplexes studied.

unwinding than the β-LNA:RNA duplex and different thermodynamic stability. The extent of structural and energetic perturbation for α-LNA modification is larger compared to βLNA modification observed in base pair step parameters (Figures S12 and S13 in the Supporting Information). The binding of α-/β-LNA strands complementary to RNA strand results in a stable LNA:RNA duplex but the extent of stabilization is higher for β-LNA modification, suggesting a better selection of β-LNA modification over α-LNA modification. A dissimilar backbone rearrangement is observed for α-/β-LNA-modified DNA strands which further support their distinct nature. Do the Hybrid Properties Depend on the Position of Modification and the Gap between Two Modifications? Previous studies on LNA-modified duplexes suggest that the changes in duplexes are dependent on the position (5′ or 3′ or center) of modification.33−35 In this section, the variations observed in hybrid properties when introducing the LNA analogues at different positions, i.e., close to 5′, center, and close to the 3′ end, are discussed. The single-modified systems LNA11, LNA12, and LNA13 are discussed in this section. The LNA11 and LNA13 have modifications near the terminal ends, and LNA12 has modification at middle position (see Figure 1). The observed variations in structural and energetic features indicate that the dissimilar nature of α-/β-LNA modifications toward affecting the DNA−RNA hybrid duplex properties. The modification at central position results in a duplex different from the one with terminal modifications for both α-/β-LNA modifications. Qualitative trends of the relative binding free energies suggest that α-/β-LNA modification at the central position stabilizes the duplex compared to the terminal modifications. In other words, the influence of LNA modification on the DNA−RNA hybrid duplex is position dependent. Similar differences can also be found in other duplexes like pure DNA and pure RNA duplexes. This subsection covers the impact of isomeric LNA modifications when modifying the deoxyribose sugars at two positions while maintaining different gaps between them. The

It is noticed that the calculated force constants are higher for βLNA-modified duplexes and smaller for α-LNA-modified duplexes than DNA−RNA hybrid duplex (Table S3 in the Supporting Information). This indicates that the β-LNAmodified duplexes are more rigid than the DNA−RNA hybrid and α-LNA-modified duplexes in all the local and global helical deformations. In other words, the deformation of the DNA− RNA hybrid duplex along its translational and rotational motions will become moderately easy after introducing α-LNA modifications, and it will be slightly stiff when introducing βLNA modifications. This indicates the opposite nature of the isomeric α-/β-LNA modifications on the stiffness of the DNA− RNA hybrid duplex, in support of the harmonic force constants and intramolecular entropies. Comparison of 3α-LNA−DNA:RNA versus 3β-LNA− DNA:RNA and α-LNA:RNA versus β-LNA:RNA duplexes. The previous studies suggest that more than three modifications are sufficient to change the duplex conformation into Atype. To understand this, the three α/β-modified duplexes are compared here. Appreciable changes are not found from the calculated intra-phosphate−phosphate (P−P) distances (Figure S9 in the Supporting Information). The partial α-LNAmodified DNA−RNA hybrid duplexes show higher helical unwinding than partial β-LNA-modified nucleic acid duplexes as observed in full LNA-modified duplexes41 (Figures S10 and S11 in the Supporting Information). The present MD simulation resulted in a 3 α-LNA-modified duplex (LNA3) structure that exhibits a larger helical unwinding compared to DNA−RNA hybrid and 3 β-LNA-modified duplex. The variations in global and local base pair step parameters suggest an altered geometrical nature for both the LNA3 duplexes. The fully modified DNA−RNA hybrid duplexes indicate that the full α-LNA modification slightly increases the intrastrand P−P distance of the modified strand and decreases the intrastrand P−P distances of the complementary strand (Figure S9). But the β-modified strand has small P−P distances compared to pure DNA−RNA hybrid and full α-LNA-modified duplex. The α-LNA:RNA duplex exhibits higher helical 5860

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of hydrogen bond lengths in the base pairs, probability distributions of backbone dihedral angles, average values of base pair step and helical parameters, time series of helical parameters, and intrastrand phosphate−phosphate distances. This material is available free of charge via the Internet at http://pubs.acs.org.

systems LNA23, LNA21, and LNA22 have 1, 2, and 3 intermediate nucleotides (gap) between two successive LNA modifications. The variations observed in the properties of these systems indicate that the effect of each modification is similar to the respective single modification, suggesting an additive nature of the modifications. The various energy and structural calculations suggest that these two isomeric α-/βLNA modifications affect the duplex peculiarly when the gap is increased between them. The β-LNA modifications stabilize the duplex with large gap between them whereas the α-LNA modification destabilizes it. It may be true that the stability of the duplex increases as the increase in gap between two modifications increases for β-LNA modification but not for αLNA modification. Future studies with various number of sequence lengths are required to confirm this. The various trends observed in sugar conformations and helicoidal parameters suggest that the gap between two modifications play a significant role in enhancing the duplex properties.

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Corresponding Author

*E-mail: [email protected]. Phone: +91-40-6653 1161. Fax: +9140-6653 1413. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS G.S. thanks Council of Scientific and Industrial Research (CSIR), India, for a senior research fellowship. U.D.P. thanks the Department of Biotechnology (Government of India) for the IYBA award. Department of Atomic Energy (Govt. of India) is acknowledged for financial assistance.

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CONCLUSIONS MD simulations have been performed to understand the change in structures, dynamics, and stability of DNA−RNA hybrid duplexes when systematically modifying its deoxyribose sugars with isomeric LNA (α-/β-LNA) modifications. The changes in structure and energetics due to these modifications were examined using various analyses, including principal component (PCA) and free energy calculations. The observed trends suggest that these two isomeric modifications (α-/βLNA) significantly affect the stability/stiffness of the DNA− RNA hybrid duplex. The binding free energies indicate that the full strand modified α-LNA:RNA and β-LNA:RNA duplexes are quite stable compared to pure DNA−RNA hybrid duplex. Some of the partial modified duplexes are also stable. The structural perturbation in the DNA−RNA hybrid duplex is high for α-LNA modification compared to β-LNA. For example, there is an unusual disparity in helical twist for α-LNA-modified duplexes compared to β-LNA-modified duplexes. Combination of the present results and previous MD results on full α-/βLNA-modified duplexes41 suggests that the β-LNA modification can mimic ribose sugar, but the α-LNA does not mimic the deoxyribose sugar. The present MD results also suggest that 3 β-LNA modifications were not sufficient to change the DNA− RNA duplex conformation into A-like. But the increasing βLNA modifications slowly turn the DNA−RNA duplex into Alike but the α-LNA modification changes the conformation instead of preserving its A/B-like conformation. The full strand modified α-LNA:RNA and β-LNA:RNA duplexes show properties distinct from pure DNA−RNA hybrid duplex. The current MD simulation results also suggest that the perturbations in DNA−RNA hybrid duplex properties are dependent on the position of modification and the gap between two modifications. The population distribution of backbone dihedral angles BI versus BII suggests that the β-LNA modification can be incorporated into the DNA strand of the DNA−RNA hybrid duplex without backbone rearrangement whereas backbone rearrangement is required in order to accommodate the α-LNA modification, resulting in unstable duplexes for some of the partial modifications.

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AUTHOR INFORMATION

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Tables of rmsf and elastic force constants, and figures of time series of root-mean-square distances, probability distributions 5861

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