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

Differential Base Stacking Interactions Induced by Trimethylene Interstrand DNA Cross-Links in the 5′-CpG-3′ and 5′-GpC-3′ Sequence Contexts

Synthetically derived trimethylene interstrand DNA cross-links have been used as surrogates for the native cross-links that arise from the 1,N 2-deoxyguanosine adducts derived from R,β-unsaturated aldehydes. The native enal-mediated cross-linking occurs in the 5′-CpG-3 ′ sequence context but not in the 5′-GpC-3 ′ sequence context. The ability of the native enal-derived 1,N 2-dG adducts to induce interstrand DNA cross-links in the 5′-CpG-3 ′ sequence as opposed to the 5′-GpC-3 ′ sequence is attributed to the destabilization of the DNA duplex in the latter sequence context. Here, we report higher accuracy solution structures of the synthetically derived trimethylene cross-links, which are refined from NMR data with the AMBER force field. When the synthetic trimethylene cross-links are placed into either the 5′-CpG-3′ or the 5′-GpC-3 ′ sequence contexts, the DNA duplex maintains B-DNA geometry with structural perturbations confined to the cross-linked base pairs. Watson-Crick hydrogen bonding is conserved throughout the duplexes. Although different from canonical B-DNA stacking, the cross-linked and the neighbor base pairs stack in the 5′-CpG-3 ′ sequence. In contrast, the stacking at the cross-linked base pairs in the 5′-GpC-3 ′ sequence is greatly perturbed. The π-stacking interactions between the crosslinked and the neighbor base pairs are reduced. This is consistent with remarkable chemical shift perturbations of the C 5 H5 and H6 nucleobase protons that shifted downfield by 0.4-0.5 ppm. In contrast

Sequence-Dependent Fluorescence of Cyanine Dyes on Microarrays

Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5′-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5′ guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5′-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling

Swarm Learning for decentralized and confidential clinical machine learning

Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning—a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine

On the coordination of La3+ by phosphatidylserine.

In a recent study by Bentz, J., D. Alford, J. Cohen, and N. Düzgünes (1988. Biophys. J. 53:593-607), La3+ was found to be more effective than Ca2+ in causing nonleaky fusion of phosphatidylserine vesicles. It was proposed that this difference in fusion efficiency may be due, in part, to a difference in coordination of the two cations. That is, Ca2+ was presumed to bind to the lipid phosphate, whereas La3+ was proposed to be coordinated by the serine carboxylate and amine. 31P and 13C NMR results presented here demonstrate that the lanthanides, Tb3+ and La3+, are coordinated by the phosphodiester and carboxylate moieties of phosphatidylserine. Tb3+-Phosphatidylserine optical experiments suggest that the serine amine does not coordinate the lanthanide below pH 10, at least not while the membrane has a net negative surface charge. Although these observations disagree with the structural details proposed by Bentz et al. (1988), they are not in conflict with their general fusion mechanism. The work presented here also demonstrates that La3+ affects the inner surface phosphodiesters differently than those on the outer surface of phosphatidylserine vesicles. The vesicles studied are of an intermediate size, having diameters on the order of 150-200 nm. The cation appears to have a more immediate effect on the packing of the crowded headgroups on the inner surface. Higher levels of bound La3+ on the outer surface may be required to induce the same changes in headgroup conformation