Interplay of LNA and 2′‑<i>O</i>‑Methyl RNA in the Structure and Thermodynamics of RNA Hybrid Systems: A Molecular Dynamics Study Using the Revised AMBER Force Field and Comparison with Experimental Results

When used in nucleic acid duplexes, locked nucleic acid (LNA) and 2′-<i>O</i>-methyl RNA residues enhance the duplex stabilities, and this makes it possible to create much better RNA aptamers to target specific molecules in cells. Thus, LNA and 2′-<i>O</i>-methyl RNA residues are finding increasingly widespread use in RNA-based therapeutics. Herein, we utilize molecular dynamics (MD) simulations and UV melting experiments to investigate the structural and thermodynamic properties of 13 nucleic acid duplexes, including full DNA, RNA, LNA, and 2′-<i>O</i>-methyl RNA duplexes as well as hybrid systems such as LNA:RNA, 2′-<i>O</i>-methyl RNA:RNA, LNA/2′-<i>O</i>-methyl RNA:RNA, and RNA/2′-<i>O</i>-methyl RNA:RNA duplexes. The MD simulations are based on a version of the Amber force field revised specifically for RNA and LNA residues. Our results indicate that LNA and 2′-<i>O</i>-methyl RNA residues have two different hybridization mechanisms when included in hybrid duplexes with RNA wherein the former underwinds while the latter overwinds the duplexes. These computational predictions are supported by X-ray structures of LNA and 2′-<i>O</i>-methyl RNA duplexes that were recently presented by different groups, and there is also good agreement with the measured thermal stabilities of the duplexes. We find out that the “underwinding” phenomenon seen in LNA and LNA:RNA hybrid duplexes happens due to expansion of the major groove widths (Mgw) of the duplexes that is associated with decrease in the slide and twist values in base-pair steps. In contrast, 2′-<i>O</i>-methyl RNA residues in RNA duplexes slightly overwind the duplexes while the backbone is forced to stay in C3′-endo. Moreover, base-pair stacking in the LNA and LNA:RNA hybrid systems is gradually reduced with the inclusion of LNA residues in the duplexes while no such effect is seen in the 2′-<i>O</i>-methyl RNA systems. Our results show how competition between base stacking and structural rigidity in these RNA hybrid systems influences structures and stabilities. Even though both LNA and 2′-<i>O</i>-methyl RNA residues have C3′-endo sugar puckering, structurally LNA residues have a frozen sugar backbone which provides entropic enhancement of stabilities while the 2′-<i>O</i>-methyl RNA residues are more flexible and maintain base stacking that is almost untouched compared to RNA. Thus, enhancement of the structural stabilities of RNA duplexes by 2′-<i>O</i>-methyl RNA modifications is smaller than for the corresponding LNA modifications. Indeed, our experimental measurements show that on average each 2′-<i>O</i>-methyl RNA and LNA substitution in a RNA duplex enhances duplex stability by 0.2 and 1.4 kcal/mol, respectively. Our computational binding free energy predictions are qualitatively in line with these results. The only exception is for the full 2′-<i>O</i>-methyl RNA duplex, which is overstabilized, implying that further force field revisions are needed. Collectively, the results presented in this paper explain the atomistic details of the structural and thermodynamic roles of LNA and 2′-<i>O</i>-methyl RNA residues in RNA hybrid duplexes, shedding light on the mechanism behind targeting endogenous micro RNA (miRNA) in order to regulate mRNA activity and inhibit gene expression in the cell

A Dynamic Structural Model of Expanded RNA CAG Repeats: A Refined X‑ray Structure and Computational Investigations Using Molecular Dynamics and Umbrella Sampling Simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, a crystal structure of r­[5′<u>UU</u>GGGC­(C<u>A</u>G)₃GUCC]₂, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti–anti and syn–anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r­(5′GCGC<u>A</u>GCGC)₂ (MS1) and r­(5′CCGC<u>A</u>GCGG)₂ (MS2). In these MD simulations, both anti–anti and syn–anti AA base pairs appear to be stable. While anti–anti AA base pairs were dynamic and sampled multiple anti–anti conformations, no syn–anti ↔ anti–anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r­[5′GGGC­(C<u>A</u>G)₃GUCC]₂, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn–anti conformation), transformed to anti–anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn–anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r­(CAG) repeats is anti–anti but can adopt syn–anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles

A Dynamic Structural Model of Expanded RNA CAG Repeats: A Refined X‑ray Structure and Computational Investigations Using Molecular Dynamics and Umbrella Sampling Simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, a crystal structure of r­[5′<u>UU</u>GGGC­(C<u>A</u>G)₃GUCC]₂, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti–anti and syn–anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r­(5′GCGC<u>A</u>GCGC)₂ (MS1) and r­(5′CCGC<u>A</u>GCGG)₂ (MS2). In these MD simulations, both anti–anti and syn–anti AA base pairs appear to be stable. While anti–anti AA base pairs were dynamic and sampled multiple anti–anti conformations, no syn–anti ↔ anti–anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r­[5′GGGC­(C<u>A</u>G)₃GUCC]₂, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn–anti conformation), transformed to anti–anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn–anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r­(CAG) repeats is anti–anti but can adopt syn–anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles

A Dynamic Structural Model of Expanded RNA CAG Repeats: A Refined X‑ray Structure and Computational Investigations Using Molecular Dynamics and Umbrella Sampling Simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, a crystal structure of r­[5′<u>UU</u>GGGC­(C<u>A</u>G)₃GUCC]₂, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti–anti and syn–anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r­(5′GCGC<u>A</u>GCGC)₂ (MS1) and r­(5′CCGC<u>A</u>GCGG)₂ (MS2). In these MD simulations, both anti–anti and syn–anti AA base pairs appear to be stable. While anti–anti AA base pairs were dynamic and sampled multiple anti–anti conformations, no syn–anti ↔ anti–anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r­[5′GGGC­(C<u>A</u>G)₃GUCC]₂, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn–anti conformation), transformed to anti–anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn–anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r­(CAG) repeats is anti–anti but can adopt syn–anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles

Hydrophobic Organic Linkers in the Self-Assembly of Small Molecule-DNA Hybrid Dimers: A Computational–Experimental Study of the Role of Linkage Direction in Product Distributions and Stabilities

Detailed computational and experimental studies reveal the crucial role that hydrophobic interactions play in the self-assembly of small molecule-DNA hybrids (SMDHs) into cyclic nanostructures. In aqueous environments, the distribution of the cyclic structures (dimers or higher-order structures) greatly depends on how well the hydrophobic surfaces of the organic cores in these nanostructures are minimized. Specifically, when the cores are attached to the 3′-ends of the DNA component strands, they can insert into the minor groove of the duplex that forms upon self-assembly, favoring the formation of cyclic dimers. However, when the cores are attached to the 5′-ends of the DNA component strands, such insertion is hindered, leading to the formation of higher-order cyclic structures. These computational insights are supported by experimental results that show clear differences in product distributions and stabilities for a broad range of organic core-linked DNA hybrids with different linkage directions and flexibilities

Enhancing the Melting Properties of Small Molecule-DNA Hybrids through Designed Hydrophobic Interactions: An Experimental-Computational Study

Detailed experimental and computational studies revealed the important role that hydrophobic interactions play in the aqueous assembly of rigid small molecule-DNA hybrid (rSMDH) building blocks into nanoscale cage and face-to-face (ff) dimeric structures. In aqueous environments, the hydrophobic surfaces of the organic cores in these nanostructures are minimized by interactions with the core in another rSMDHs, with the bases in the attached DNA strands, and/or with the base pairs in the final assembled structures. In the case that the hydrophobic surfaces of the cores could not be properly isolated in the assembly process, an ill-defined network results instead of dimers, even at low concentration of DNA. In contrast, if ff dimers can be formed with good minimization of the exposed hydrophobic surfaces of the cores, they are highly stable structures with enhanced melting temperatures and cooperative melting behavior

Hydrophobic Organic Linkers in the Self-Assembly of Small Molecule-DNA Hybrid Dimers: A Computational–Experimental Study of the Role of Linkage Direction in Product Distributions and Stabilities

Detailed computational and experimental studies reveal the crucial role that hydrophobic interactions play in the self-assembly of small molecule-DNA hybrids (SMDHs) into cyclic nanostructures. In aqueous environments, the distribution of the cyclic structures (dimers or higher-order structures) greatly depends on how well the hydrophobic surfaces of the organic cores in these nanostructures are minimized. Specifically, when the cores are attached to the 3′-ends of the DNA component strands, they can insert into the minor groove of the duplex that forms upon self-assembly, favoring the formation of cyclic dimers. However, when the cores are attached to the 5′-ends of the DNA component strands, such insertion is hindered, leading to the formation of higher-order cyclic structures. These computational insights are supported by experimental results that show clear differences in product distributions and stabilities for a broad range of organic core-linked DNA hybrids with different linkage directions and flexibilities

A Dynamic Structural Model of Expanded RNA CAG Repeats: A Refined X‑ray Structure and Computational Investigations Using Molecular Dynamics and Umbrella Sampling Simulations

One class of functionally important RNA is repeating transcripts that cause disease through various mechanisms. For example, expanded CAG repeats can cause Huntington’s and other disease through translation of toxic proteins. Herein, a crystal structure of r­[5′<u>UU</u>GGGC­(C<u>A</u>G)₃GUCC]₂, a model of CAG expanded transcripts, refined to 1.65 Å resolution is disclosed that shows both anti–anti and syn–anti orientations for 1 × 1 nucleotide AA internal loops. Molecular dynamics (MD) simulations using AMBER force field in explicit solvent were run for over 500 ns on the model systems r­(5′GCGC<u>A</u>GCGC)₂ (MS1) and r­(5′CCGC<u>A</u>GCGG)₂ (MS2). In these MD simulations, both anti–anti and syn–anti AA base pairs appear to be stable. While anti–anti AA base pairs were dynamic and sampled multiple anti–anti conformations, no syn–anti ↔ anti–anti transformations were observed. Umbrella sampling simulations were run on MS2, and a 2D free energy surface was created to extract transformation pathways. In addition, an explicit solvent MD simulation over 800 ns was run on r­[5′GGGC­(C<u>A</u>G)₃GUCC]₂, which closely represents the refined crystal structure. One of the terminal AA base pairs (syn–anti conformation), transformed to anti–anti conformation. The pathway followed in this transformation was the one predicted by umbrella sampling simulations. Further analysis showed a binding pocket near AA base pairs in syn–anti conformations. Computational results combined with the refined crystal structure show that global minimum conformation of 1 × 1 nucleotide AA internal loops in r­(CAG) repeats is anti–anti but can adopt syn–anti depending on the environment. These results are important to understand RNA dynamic-function relationships and to develop small molecules that target RNA dynamic ensembles

Computational Investigation of RNA CUG Repeats Responsible for Myotonic Dystrophy 1

Myotonic Dystrophy 1 (DM1) is a genetic disease caused by expansion of CTG repeats in DNA. Once transcribed, these repeats form RNA hairpins with repeating 1×1 nucleotide UU internal loop motifs, r­(C<u>U</u>G)_n, which attract muscleblind-like 1 (MBNL1) protein leading to the disease. In DM1 C<u>U</u>G can be repeated thousands of times, so these structures are intractable to characterization using structural biology. However, inhibition of MBNL1-r­(C<u>U</u>G)_n binding requires a detailed analysis of the 1×1 UU internal loops. In this contribution we employ regular and umbrella sampling molecular dynamics (MD) simulations to describe the structural and thermodynamic properties of 1×1 UU internal loops. Calculations were run on a reported crystal structure and a designed system, which mimics an infinitely long RNA molecule with continuous C<u>U</u>G repeats. Two-dimensional (2D) potential of mean force (PMF) surfaces were created by umbrella sampling, and the discrete path sampling (DPS) method was utilized to investigate the energy landscape of 1×1 UU RNA internal loops, revealing that 1×1 UU base pairs are dynamic and strongly prefer the <i>anti</i>–<i>anti</i> conformation. Two 2D PMF surfaces were calculated for the 1×1 UU base pairs, revealing several local minima and three <i>syn</i>–<i>anti</i> ↔ <i>anti</i>–<i>anti</i> transformation pathways. Although at room temperature the <i>syn</i>–<i>anti</i> ↔ <i>anti</i>–<i>anti</i> transformation is not observed on the MD time scale, one of these pathways dominates the dynamics of the 1×1 UU base pairs in temperature jump MD simulations. This mechanism has now been treated successfully using the DPS approach. Our results suggest that local minima predicted by umbrella sampling calculations could be stabilized by small molecules, which is of great interest for future drug design. Furthermore, distorted GC/CG conformations may be important in understanding how MBNL1 binds to RNA C<u>U</u>G repeats. Hence we provide new insight into the dynamic roles of RNA loops and their contributions to presently incurable diseases