Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

Fly casting with ligand sliding and orientational selection supporting complex formation of a GPCR and a middle sized flexible molecule.

A GA-guided multidimensional virtual-system coupled molecular dynamics (GA-mD-VcMD) simulation was conducted to elucidate binding mechanisms of a middle-sized flexible molecule, bosentan, to a GPCR protein, human endothelin receptor type B (hETB). GA-mD-VcMD is a generalized ensemble method that produces a free-energy landscape of the ligand-receptor binding by searching large-scale motions accompanied with stable maintenance of the fragile cell-membrane structure. All molecular components (bosentan, hETB, membrane, and solvent) were represented with an all-atom model. Then sampling was conducted from conformations where bosentan was distant from the binding site in the hETB binding pocket. The deepest basin in the resultant free-energy landscape was assigned to native-like complex conformation. The following binding mechanism was inferred. First, bosentan fluctuating randomly in solution is captured using a tip region of the flexible N-terminal tail of hETB via nonspecific attractive interactions (fly casting). Bosentan then slides occasionally from the tip to the root of the N-terminal tail (ligand-sliding). During this sliding, bosentan passes the gate of the binding pocket from outside to inside of the pocket with an accompanying rapid reduction of the molecular orientational variety of bosentan (orientational selection). Last, in the pocket, ligand-receptor attractive native contacts are formed. Eventually, the native-like complex is completed. The bosentan-captured conformations by the tip-region and root-region of the N-terminal tail correspond to two basins in the free-energy landscape. The ligand-sliding corresponds to overcoming of a free-energy barrier between the basins.

HNF1A POU Domain Mutations Found in Japanese Liver Cancer Patients Cause Downregulation of HNF4A Promoter Activity with Possible Disruption in Transcription Networks.

Hepatocyte nuclear factor 1A (HNF1A) is the master regulator of liver homeostasis and organogenesis and regulates many aspects of hepatocyte functions. It acts as a tumor suppressor in the liver, evidenced by the increased proliferation in HNF1A knockout (KO) hepatocytes. Hence, we postulated that any loss-of-function variation in the gene structure or composition (mutation) could trigger dysfunction, including disrupted transcriptional networks in liver cells. From the International Cancer Genome Consortium (ICGC) database of cancer genomes, we identified several HNF1A mutations located in the functional Pit-Oct-Unc (POU) domain. In our biochemical analysis, we found that the HNF1A POU-domain mutations Y122C, R229Q and V259F suppressed HNF4A promoter activity and disrupted the binding of HNF1A to its target HNF4A promoter without any effect on the nuclear localization. Our results suggest that the decreased transcriptional activity of HNF1A mutants is due to impaired DNA binding. Through structural simulation analysis, we found that a V259F mutation was likely to affect DNA interaction by inducing large conformational changes in the N-terminal region of HNF1A. The results suggest that POU-domain mutations of HNF1A downregulate HNF4A gene expression. Therefore, to mimic the HNF1A mutation phenotype in transcription networks, we performed siRNA-mediated knockdown (KD) of HNF4A. Through RNA-Seq data analysis for the HNF4A KD, we found 748 differentially expressed genes (DEGs), of which 311 genes were downregulated (e.g., HNF1A, ApoB and SOAT2) and 437 genes were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed that the DEGs were involved in several signaling pathways (e.g., lipid and cholesterol metabolic pathways). Protein-protein network analysis suggested that the downregulated genes were related to lipid and cholesterol metabolism pathways, which are implicated in hepatocellular carcinoma (HCC) development. Our study demonstrates that mutations of HNF1A in the POU domain result in the downregulation of HNF1A target genes, including HNF4A, and this may trigger HCC development through the disruption of HNF4A-HNF1A transcriptional networks.

Extended ensemble simulations of a SARS-CoV-2 nsp1-5'-UTR complex.

Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 180-residue protein that blocks translation of host mRNAs in SARS-CoV-2-infected cells. Although it is known that SARS-CoV-2's own RNA evades nsp1's host translation shutoff, the molecular mechanism underlying the evasion was poorly understood. We performed an extended ensemble molecular dynamics simulation to investigate the mechanism of the viral RNA evasion. Simulation results suggested that the stem loop structure of the SARS-CoV-2 RNA 5'-untranslated region (SL1) binds to both nsp1's N-terminal globular region and intrinsically disordered region. The consistency of the results was assessed by modeling nsp1-40S ribosome structure based on reported nsp1 experiments, including the X-ray crystallographic structure analysis, the cryo-EM electron density map, and cross-linking experiments. The SL1 binding region predicted from the simulation was open to the solvent, yet the ribosome could interact with SL1. Cluster analysis of the binding mode and detailed analysis of the binding poses suggest residues Arg124, Lys47, Arg43, and Asn126 may be involved in the SL1 recognition mechanism, consistent with the existing mutational analysis.

Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2.

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

The N-terminal Tails of Histones H2A and H2B Adopt Two Distinct Conformations in the Nucleosome with Contact and Reduced Contact to DNA.

The nucleosome comprises two histone dimers of H2A-H2B and one histone tetramer of (H3-H4)2, wrapped around by ~145 bp of DNA. Detailed core structures of nucleosomes have been established by X-ray and cryo-EM, however, histone tails have not been visualized. Here, we have examined the dynamic structures of the H2A and H2B tails in 145-bp and 193-bp nucleosomes using NMR, and have compared them with those of the H2A and H2B tail peptides unbound and bound to DNA. Whereas the H2A C-tail adopts a single but different conformation in both nucleosomes, the N-tails of H2A and H2B adopt two distinct conformations in each nucleosome. To clarify these conformations, we conducted molecular dynamics (MD) simulations, which suggest that the H2A N-tail can locate stably in either the major or minor grooves of nucleosomal DNA. While the H2B N-tail, which sticks out between two DNA gyres in the nucleosome, was considered to adopt two different orientations, one toward the entry/exit side and one on the opposite side. Then, the H2A N-tail minor groove conformation was obtained in the H2B opposite side and the H2B N-tail interacts with DNA similarly in both sides, though more varied conformations are obtained in the entry/exit side. Collectively, the NMR findings and MD simulations suggest that the minor groove conformer of the H2A N-tail is likely to contact DNA more strongly than the major groove conformer, and the H2A N-tail reduces contact with DNA in the major groove when the H2B N-tail is located in the entry/exit side.

Free-Energy Calculation of Ribonucleic Inosines and Its Application to Nearest-Neighbor Parameters.

Can current simulations quantitatively predict the stability of ribonucleic acids (RNAs)? In this research, we apply a free-energy perturbation simulation of RNAs containing inosine, a modified ribonucleic base, to the derivation of RNA nearest-neighbor parameters. A parameter set derived solely from 30 simulations was used to predict the free-energy difference of the RNA duplex with a mean unbiased error of 0.70 kcal/mol, which is a level of accuracy comparable to that obtained with parameters derived from 25 experiments. We further show that the error can be lowered to 0.60 kcal/mol by combining the simulation-derived free-energy differences with experimentally measured differences. This protocol can be used as a versatile method for deriving nearest-neighbor parameters of RNAs with various modified bases.

A common allosteric mechanism regulates homeostatic inactivation of auxin and gibberellin.

Allosteric regulation is protein activation by effector binding at a site other than the active site. Here, we show via X-ray structural analysis of gibberellin 2-oxidase 3 (GA2ox3), and auxin dioxygenase (DAO), that such a mechanism maintains hormonal homeostasis in plants. Both enzymes form multimers by interacting via GA4 and indole-3-acetic acid (IAA) at their binding interface. Via further functional analyses we reveal that multimerization of these enzymes gradually proceeds with increasing GA4 and IAA concentrations; multimerized enzymes have higher specific activities than monomer forms, a system that should favour the maintenance of homeostasis for these phytohormones. Molecular dynamic analysis suggests a possible mechanism underlying increased GA2ox3 activity by multimerization-GA4 in the interface of oligomerized GA2ox3s may be able to enter the active site with a low energy barrier. In summary, homeostatic systems for maintaining GA and IAA levels, based on a common allosteric mechanism, appear to have developed independently.

Large-Scale Parallel Implementation of Hartree-Fock Exchange Energy on Real-Space Grids Using 3D-Parallel Fast Fourier Transform.

Two types of implementation of the Hartree-Fock (HF) exchange energy were developed with the real-space grid approach for the purpose of achieving high efficiency in the parallel execution of the hybrid exchange functional in the density functional theory. First, a parallel implementation of the three-dimensional fast Fourier transform (FFT), referred to as PFFT, was adapted to solve the Poisson equations for the electrostatic potentials of the densities of the orbital pairs. In the other approach, the Poisson equations were solved iteratively through the conjugate gradient (CG) procedures where the operation of Laplacian was parallelized by the domain decomposition scheme. Comparison of the parallel performances for the exchange energy calculation was made between these two approaches, and it was revealed that the calculation with the FFT method is faster than that with CG. The method with FFT is more advantageous than CG because a larger bandwidth can be made available in the collective message passing interface communication associated with the parallel execution of FFT. We also implemented the projection operator to circumvent the laborious calculation of the exchange energy at every self-consistent field step, which made a significant contribution to expedite the convergence. To assess the accuracy of our implementation, the association energies of a hydrated ion were computed, which showed excellent agreement with those given by the Gaussian 09 program employing sophisticated basis sets.

Correction: Free energy profiles for unwrapping the outer superhelical turn of nucleosomal DNA.

[This corrects the article DOI: 10.1371/journal.pcbi.1006024.].

Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation.

Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

Role of the Photosystem II as an Environment in the Oxidation Free Energy of the Mn Cluster from S1 to S2.

The manganese cluster (CaMn4O5) in the photosystem II (PSII) is the reaction center of the light-driven oxidation reaction, which generates the molecular oxygen. In this paper, we address the issue of the effect of the environment on the free energy associated with the oxidation of the Mn cluster in S1 state by conducting the large-scale quantum mechanical/molecular mechanical simulations, which involve the whole of the PSII monomer. It was found by the simulations at the level of the B3LYP functional that the environment surrounding the Mn cluster reduces the vertical oxidation free energy Δµvrt by 64.8 kcal/mol. A decomposition analysis of the free energy Δµvrt revealed that the system composed of peptide chains, ligands, lipids, and potassium ions contributes to lowering of Δµvrt by -98.0 kcal/mol, whereas the solvent water makes an opposite contribution of 38.9 kcal/mol. Reduction of the vertical oxidation free energy directly leads to the lowering of the activation free energy ΔGac for the electron transfer reaction from the Mn cluster in S1 state to the neighboring Tyrz+. Consequently, the electron transfer rate was found to be enhanced by a factor of 1012 by virtue of the influence of the environment.

Critical Roles for Coiled-Coil Dimers of Butyrophilin 3A1 in the Sensing of Prenyl Pyrophosphates by Human Vγ2Vδ2 T Cells.

Vγ2Vδ2 T cells play important roles in human immunity to pathogens and tumors. Their TCRs respond to the sensing of isoprenoid metabolites, such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and isopentenyl pyrophosphate, by butyrophilin (BTN) 3A1. BTN3A1 is an Ig superfamily protein with extracellular IgV/IgC domains and intracellular B30.2 domains that bind prenyl pyrophosphates. We have proposed that intracellular α helices form a coiled-coil dimer that functions as a spacer for the B30.2 domains. To test this, five pairs of anchor residues were mutated to glycine to destabilize the coiled-coil dimer. Despite maintaining surface expression, BTN3A1 mutagenesis either abrogated or decreased stimulation by (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate. BTN3A2 and BTN3A3 proteins and orthologs in alpacas and dolphins are also predicted to have similar coiled-coil dimers. A second short coiled-coil region dimerizes the B30.2 domains. Molecular dynamics simulations predict that mutation of a conserved tryptophan residue in this region will destabilize the dimer, explaining the loss of stimulation by BTN3A1 proteins with this mutation. The juxtamembrane regions of other BTN/BTN-like proteins with B30.2 domains are similarly predicted to assume α helices, with many predicted to form coiled-coil dimers. An exon at the end of this region and the exon encoding the dimerization region for B30.2 domains are highly conserved. We propose that coiled-coil dimers function as rod-like helical molecular spacers to position B30.2 domains, as interaction sites for other proteins, and as dimerization regions to allow sensing by B30.2 domains. In these ways, the coiled-coil domains of BTN3A1 play critical roles for its function.

The binding affinity of uncharged aromatic solutes for negatively charged resins is enhanced by cations via cation-π interactions: The case of sodium ion and arginine.

Salt solutions are widely used as eluents for ion-exchange chromatography. In general, salts reduce the retention of applied solutes on ion-exchange columns via electrostatic screening effects. The reverse phenomenon, namely, salt-enhanced retention, has not been reported. Here, we report that cations, including arginine, guanidine and sodium ions, enhance the retention of uncharged aromatic solutes on a cation-exchange resin, i.e., a negatively charged resin, with carboxyl groups, where we used alkyl gallates as model uncharged aromatic solutes and a carboxymethyl agarose gel (CM Sepharose) as a model negatively charged resin. Enhancement of retention was observed at concentrations of tens of millimolar of the salts, in which arginine hydrochloride was more effective than guanidinium salts and NaCl. Similar trends were observed for other phenolic compounds, including phenol and 4-hydroxybenzyl alcohol. Molecular dynamics simulations showed that the binding free energy between the alkyl gallate molecule and the CM Sepharose resin ligand molecule increased with increasing salt concentration. The increase in binding free energy caused by the salts was accounted for by the binding of the salt cations to the aromatic moiety of the alkyl gallate via cation-π interactions, leading to attenuation of intrinsic repulsive interactions between the ligand carboxyl group and the alkyl gallate aromatic moiety. Therefore, the salt-enhanced retention of the uncharged aromatic solutes on the negatively charged resins was ascribable to the increase in binding free energy induced by the cation-π interactions. This unique reverse phenomenon of the effect of salts on solute retention indicates the importance of cation-π interactions in ion-exchange chromatography. This phenomenon can be used for selective chromatographic separation of aromatic solutes, including organic solutes, proteins and nucleic acids.

Free energy profile for unwrapping outer superhelical turn of CENP-A nucleosome.

Eukaryotic genome is packaged in a nucleus in the form of chromatin. The fundamental structural unit of the chromatin is the protein-DNA complex, nucleosome, where DNA of about 150 bp is wrapped around a histone core almost twice. In cellular processes such as gene expression, DNA repair and duplication, the nucleosomal DNA has to be unwrapped. Histone proteins have their variants, indicating there are a variety of constitutions of nucleosomes. These different constitutions are observed in different cellular processes. To investigate differences among nucleosomes, we calculated free energy profiles for unwrapping the outer superhelical turn of CENP-A nucleosome and compared them with those of the canonical nucleosome. The free energy profiles for CENP-A nucleosome suggest that CENP-A nucleosome is the most stable when 16 to 22 bps are unwrapped in total whereas the canonical nucleosome is the most stable when it is fully wrapped. This indicates that the flexible conformation of CENP-A nucleosome is ready to provide binding sites for the structural integrity of the centromere.

Estimating Energy Parameters for RNA Secondary Structure Predictions Using Both Experimental and Computational Data.

Computational RNA secondary structure prediction depends on a large number of nearest-neighbor free-energy parameters, including 10 parameters for Watson-Crick stacked base pairs that were estimated from experimental measurements of the free energies of 90 RNA duplexes. These experimental data are provided by time-consuming and cost-intensive experiments. In contrast, various modified nucleotides in RNAs, which would affect not only their structures but also functions, have been found, and rapid determination of energy parameters for a such modified nucleotides is needed. To reduce the high cost of determining energy parameters, we propose a novel method to estimate energy parameters from both experimental and computational data, where the computational data are provided by a recently developed molecular dynamics simulation protocol. We evaluate our method for Watson-Crick stacked base pairs, and show that parameters estimated from 10 experimental data items and 10 computational data items can predict RNA secondary structures with accuracy comparable to that using conventional parameters. The results indicate that the combination of experimental free-energy measurements and molecular dynamics simulations is capable of estimating the thermodynamic properties of RNA secondary structures at lower cost.

Performance evaluation of the zero-multipole summation method in modern molecular dynamics software.

The zero-multiple summation method (ZMM) is a cutoff-based method for calculating electrostatic interactions in molecular dynamics simulations, utilizing an electrostatic neutralization principle as a physical basis. Since the accuracies of the ZMM have been revealed to be sufficient in previous studies, it is highly desirable to clarify its practical performance. In this paper, the performance of the ZMM is compared with that of the smooth particle mesh Ewald method (SPME), where the both methods are implemented in molecular dynamics software package GROMACS. Extensive performance comparisons against a highly optimized, parameter-tuned SPME implementation are performed for various-sized water systems and two protein-water systems. We analyze in detail the dependence of the performance on the potential parameters and the number of CPU cores. Even though the ZMM uses a larger cutoff distance than the SPME does, the performance of the ZMM is comparable to or better than that of the SPME. This is because the ZMM does not require a time-consuming electrostatic convolution and because the ZMM gains short neighbor-list distances due to the smooth damping feature of the pairwise potential function near the cutoff length. We found, in particular, that the ZMM with quadrupole or octupole cancellation and no damping factor is an excellent candidate for the fast calculation of electrostatic interactions. © 2018 Wiley Periodicals, Inc.

Salt-dependent elution of uncharged aromatic solutes in ion-exchange chromatography.

Ion-exchange chromatography and multimodal ion-exchange chromatography are widely used for the separation of small molecules, peptides and proteins. Salts generally attenuate the electrostatic interactions between charged moieties of solutes and those of resins through electrostatic screening. However, little is known about how salts affect the interaction between the uncharged moieties of the solutes, such as aromatic moieties, and the charged moieties of the resins. In this study, we used alkyl gallates as model aromatic solutes to investigate the interaction mechanism of aromatic moieties with multimodal and conventional ion-exchange resins. Interestingly, alkyl gallates retained by these resins were readily eluted from the columns by the addition of 0.01-1 M NaCl, even though the alkyl gallates used contained no charged group. Molecular dynamics (MD) simulations were performed to understand the mechanism of these interactions. The MD simulation with a conventional force field showed that 1 M NaCl enhances the binding of an alkyl gallate molecule to the ligand, which contradicts the experimental results. Thus, we modified the force field to express a cation-π interaction between sodium ions and aromatic moieties, which successfully reproduced the experimental results at 1 M, suggesting that the cation-π interaction between sodium ions and aromatic moieties plays a crucial role in reducing the binding affinity of alkyl gallates for the ligands. These results provide new information indicating that aromatic moieties, including the aromatic residues of proteins and nucleobases of nucleic acids, favorably interact with multimodal and conventional ion-exchange resins and that cations, such as sodium ions, contribute to attenuating the binding of aromatic moieties to the ligands.

Free energy profiles for unwrapping the outer superhelical turn of nucleosomal DNA.

The eukaryotic genome is packaged into a nucleus in the form of chromatin. The fundamental structural unit of chromatin is a protein-DNA complex, the nucleosome, where 146 or 147 base pairs of DNA wrap 1.75 times around a histone core. To function in cellular processes, however, nucleosomal DNA must be unwrapped. Although this unwrapping has been experimentally investigated, details of the process at an atomic level are not yet well understood. Here, we used molecular dynamics simulation with an enhanced sampling method to calculate the free energy profiles for unwrapping the outer superhelical turn of nucleosomal DNA. A free energy change of about 11.5 kcal/mol for the unwrapping agrees well with values obtained in single molecule experiments. This simulation revealed a variety of conformational states, indicating there are many potential paths to outer superhelicdal turn unwrapping, but the dominant path is likely asymmetric. At one end of the DNA, the first five bps unwrap, after which a second five bps unwrap at the same end with no increase in free energy. The unwrapping then starts at the other end of the DNA, where 10 bps are unwrapped. During further unwrapping of 15 bps, the unwrapping advances at one of the ends, after which the other end of the DNA unwraps to complete the unwrapping of the outer superhelical turn. These results provide insight into the construction, disruption, and repositioning of nucleosomes, which are continuously ongoing during cellular processes.

Capturing alternative secondary structures of RNA by decomposition of base-pairing probabilities.

BACKGROUND: It is known that functional RNAs often switch their functions by forming different secondary structures. Popular tools for RNA secondary structures prediction, however, predict the single 'best' structures, and do not produce alternative structures. There are bioinformatics tools to predict suboptimal structures, but it is difficult to detect which alternative secondary structures are essential. RESULTS: We proposed a new computational method to detect essential alternative secondary structures from RNA sequences by decomposing the base-pairing probability matrix. The decomposition is calculated by a newly implemented software tool, RintW, which efficiently computes the base-pairing probability distributions over the Hamming distance from arbitrary reference secondary structures. The proposed approach has been demonstrated on ROSE element RNA thermometer sequence and Lysine RNA ribo-switch, showing that the proposed approach captures conformational changes in secondary structures. CONCLUSIONS: We have shown that alternative secondary structures are captured by decomposing base-paring probabilities over Hamming distance. Source code is available from http://www.ncRNA.org/RintW .