Ilyas Yildirim

Benchmarking AMBER Force Fields for RNA: Comparisons to NMR Spectra for Single-Stranded r(GACC) Are Improved by Revised χ Torsions

Accurately modeling unpaired regions of RNA is important for predicting structure, dynamics, and thermodynamics of folded RNA. Comparisons between NMR data and molecular dynamics simulations provide a test of force fields used for modeling. Here, NMR spectroscopy, including NOESY, 1H–31P HETCOR, DQF-COSY, and TOCSY, was used to determine conformational preferences for single-stranded GACC RNA. The spectra are consistent with a conformational ensemble containing major and minor A-form-like structures. In a series of 50 ns molecular dynamics (MD) simulations with the AMBER99 force field in explicit solvent, initial A-form-like structures rapidly evolve to disordered conformations. A set of 50 ns simulations with revised χ torsions (AMBER99χ force field) gives two primary conformations, consistent with the NMR spectra. A single 1.9 μs MD simulation with the AMBER99χ force field showed that the major and minor conformations are retained for almost 68% of the time in the first 700 ns, with multiple transformations from A-form to non-A-form conformations. For the rest of the simulation, random-coil structures and a stable non-A-form conformation inconsistent with NMR spectra were seen. Evidently, the AMBER99χ force field improves structural predictions for single-stranded GACC RNA compared to the AMBER99 force field, but further force field improvements are needed.

Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1.

Revision of AMBER Torsional Parameters for RNA Improves Free Energy Predictions for Tetramer Duplexes with GC and iGiC Base Pairs

All-atom force fields are important for predicting thermodynamic, structural, and dynamic properties of RNA. In this paper, results are reported for thermodynamic integration calculations of free energy differences of duplex formation when CG pairs in the RNA duplexes r(CCGG)2, r(GGCC)2, r(GCGC)2, and r(CGCG)2 are replaced by isocytidine–isoguanosine (iCiG) pairs. Agreement with experiment was improved when ε/ζ, α/γ, β, and χ torsional parameters in the AMBER99 force field were revised on the basis of quantum mechanical calculations. The revised force field, AMBER99TOR, brings free energy difference predictions to within 1.3, 1.4, 2.3, and 2.6 kcal/mol at 300 K, respectively, compared to experimental results for the thermodynamic cycles of CCGG → iCiCiGiG, GGCC → iGiGiCiC, GCGC → iGiCiGiC, and CGCG → iCiGiCiG. In contrast, unmodified AMBER99 predictions for GGCC → iGiGiCiC and GCGC → iGiCiGiC differ from experiment by 11.7 and 12.6 kcal/mol, respectively. In order to test the dynamic stability of the above duplexes with AMBER99TOR, four individual 50 ns molecular dynamics (MD) simulations in explicit solvent were run. All except r(CCGG)2 retained A-form conformation for ≥82% of the time. This is consistent with NMR spectra of r(iGiGiCiC)2, which reveal an A-form conformation. In MD simulations, r(CCGG)2 retained A-form conformation 52% of the time, suggesting that its terminal base pairs may fray. The results indicate that revised backbone parameters improve predictions of RNA properties and that comparisons to measured sequence dependent thermodynamics provide useful benchmarks for testing force fields and computational methods.

Testing the Nearest Neighbor Model for Canonical RNA Base Pairs: Revision of GU Parameters

Thermodynamic parameters for GU pairs are important for predicting the secondary structures of RNA and for finding genomic sequences that code for structured RNA. Optical melting curves were measured for 29 RNA duplexes with GU pairs to improve nearest neighbor parameters for predicting stabilities of helixes. The updated model eliminates a prior penalty assumed for terminal GU pairs. Six additional duplexes with the 5′GG/3′UU motif were added to the single representation in the previous database. This revises the ΔG°37 for the 5′GG/3′UU motif from an unfavorable 0.5 kcal/mol to a favorable −0.2 kcal/mol. Similarly, the ΔG°37 for the 5′UG/3′GU motif changes from 0.3 to −0.6 kcal/mol. The correlation coefficients between predicted and experimental ΔG°37, ΔH°, and ΔS° for the expanded database are 0.95, 0.89, and 0.87, respectively. The results should improve predictions of RNA secondary structure.

Allosteric transcriptional regulation via changes in the overall topology of the core promoter

Transcription factor shape-shifts DNA Controlling when a gene is expressed or repressed is vital for cellular metabolism and development. In Escherichia coli, a copper-sensing transcription factor, CueR, can repress or activate expression when bound at exactly the same site in the gene promoter. Philips et al. used x-ray crystallography to show that CueR dramatically changes the topology of the DNA upon binding copper but does not affect protein-DNA contacts. The DNA shape change allows RNA polymerase to bind the promoter and transcribe the gene. Science, this issue p. 877 A transcription factor activates gene expression by dramatically changing the shape of promotor DNA. Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-form DNA–like conformation. These factors switch on and off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.

Zinc porphyrin: A fluorescent acceptor in studies of Zn-cytochrome c unfolding by fluorescence resonance energy transfer

FRET between the zinc porphyrin (ZnP) chromophore in zinc-substituted cytochrome c (Zn-cyt c) and an Alexa Fluor dye attached to specific surface sites was used to characterize Zn-cyt c unfolding. The use of ZnP as a fluorescent acceptor eliminates the need to doubly label the protein with exogenous dyes to perform FRET experiments in which both donor and acceptor fluorescence is monitored. The requirement for attachment of only one dye also minimizes perturbation to the protein. This sensitive technique allowed for the determination of distances between the label placed at six different sites and ZnP through a range of denaturant concentrations. Fitting of the data to a three-state model provides distances in the unfolding intermediate. The use of ZnP as a fluorescent acceptor of energy in FRET has a significant potential for application to a range of other systems including heme-binding proteins and proteins to which a covalently attached heme tag may be added.

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.

Optimization of an AMBER force field for the artificial nucleic acid, LNA, and benchmarking with NMR of L(CAAU).

Locked Nucleic Acids (LNAs) are RNA analogues with an O2′-C4′ methylene bridge which locks the sugar into a C3′-endo conformation. This enhances hybridization to DNA and RNA, making LNAs useful in microarrays and potential therapeutics. Here, the LNA, L(CAAU), provides a simplified benchmark for testing the ability of molecular dynamics (MD) to approximate nucleic acid properties. LNA χ torsions and partial charges were parametrized to create AMBER parm99_LNA. The revisions were tested by comparing MD predictions with AMBER parm99 and parm99_LNA against a 200 ms NOESY NMR spectrum of L(CAAU). NMR indicates an A-Form equilibrium ensemble. In 3000 ns simulations starting with an A-form structure, parm99_LNA and parm99 provide 66% and 35% agreement, respectively, with NMR NOE volumes and 3J-couplings. In simulations of L(CAAU) starting with all χ torsions in a syn conformation, only parm99_LNA is able to repair the structure. This implies methods for parametrizing force fields for nucleic acid mimics can reasonably approximate key interactions and that parm99_LNA will improve reliability of MD studies for systems with LNA. A method for approximating χ population distribution on the basis of base to sugar NOEs is also introduced.

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(CUG)n, which attract muscleblind-like 1 (MBNL1) protein leading to the disease. In DM1 CUG can be repeated thousands of times, so these structures are intractable to characterization using structural biology. However, inhibition of MBNL1-r(CUG)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 CUG 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 anti–anti conformation. Two 2D PMF surfaces were calculated for the 1×1 UU base pairs, revealing several local minima and three syn–anti ↔ anti–anti transformation pathways. Although at room temperature the syn–anti ↔ anti–anti 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 CUG repeats. Hence we provide new insight into the dynamic roles of RNA loops and their contributions to presently incurable diseases.

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.