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Singlet fission (SF), an exciton-doubling process observed in certain molecular semiconductors where two triplet excitons are generated from one singlet exciton, requires correctly tuned intermolecular coupling to allow separation of the two triplets to different molecular units. We explore this using DNA-encoded assembly of SF-capable pentacenes into discrete π-stacked constructs of defined size and geometry. Precise structural control is achieved via a combination of the DNA duplex formation between complementary single-stranded DNA and the local molecular geometry that directs the SF chromophores into a stable and predictable slip-stacked configuration, as confirmed by molecular dynamics (MD) modeling. Transient electron spin resonance spectroscopy revealed that within these DNA-assembled pentacene stacks, SF evolves via a bound triplet pair quintet state, which subsequently converts into free triplets. SF evolution via a long-lived quintet state sets specific requirements on intermolecular coupling, rendering the quintet spectrum and its zero-field-splitting parameters highly sensitive to intermolecular geometry. We have found that the experimental spectra and zero-field-splitting parameters are consistent with a slight systematic strain relative to the MD-optimized geometry. Thus, the transient electron spin resonance analysis is a powerful tool to test and refine the MD-derived structure models. DNA-encoded assembly of coupled semiconductor molecules allows controlled construction of electronically functional structures, but brings with it significant dynamic and polar disorders. Our findings here of efficient SF through quintet states demonstrate that these conditions still allow efficient and controlled semiconductor operation and point toward future opportunities for constructing functional optoelectronic systems.

Acknowledgements: We acknowledge funding from the University of Cambridge Ernest Oppenheimer Fund [PYC], the Winton Programme for the Physics of Sustainability [PYC, RC-G], the European Research Council under the European Union's Horizon 2020 research and innovation programme [grant 803326; RC-G]. JAJ is a Junior Research Fellow at King's College. This work was performed using resources provided by the Cambridge Tier-2 system operated by the University of Cambridge Research Computing Service funded by EPSRC Tier-2 capital grant EP/P020259/1 [RC-G, JAJ, AR]. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript., Intracellular condensates are highly multi-component systems in which complex phase behaviour can ensue, including the formation of architectures comprising multiple immiscible condensed phases. Relying solely on physical intuition to manipulate such condensates is difficult because of the complexity of their composition, and systematically learning the underlying rules experimentally would be extremely costly. We address this challenge by developing a computational approach to design pairs of protein sequences that result in well-separated multilayered condensates and elucidate the molecular origins of these compartments. Our method couples a genetic algorithm to a residue-resolution coarse-grained protein model. We demonstrate that we can design protein partners to form multiphase condensates containing naturally occurring proteins, such as the low-complexity domain of hnRNPA1 and its mutants, and show how homo- and heterotypic interactions must differ between proteins to result in multiphasicity. We also show that in some cases the specific pattern of amino-acid residues plays an important role. Our findings have wide-ranging implications for understanding and controlling the organisation, functions and material properties of biomolecular condensates., We acknowledge funding from the University of Cambridge Ernest Oppenheimer Fund [PYC], the Winton Programme for the Physics of Sustainability [PYC, RC-G], the European Research Council under the European Union’s Horizon 2020 research and innovation programme [grant 803326; RC-G]. JAJ is a Junior Research Fellow at King’s College. This work was performed using resources provided by the Cambridge Tier-2 system operated by the University of Cambridge Research Computing Service funded by EPSRC Tier-2 capital grant EP/P020259/1 [RC-G, JAJ, AR].

Multiphasic architectures are found ubiquitously in biomolecular condensates and are thought to have important implications for the organisation of multiple chemical reactions within the same compartment. Many of these multiphasic condensates contain RNA in addition to proteins. Here, we investigate the importance of different interactions in multiphasic condensates comprising two different proteins and RNA using computer simulations with a residue-resolution coarse-grained model of proteins and RNA. We find that in multilayered condensates containing RNA in both phases, protein-RNA interactions dominate, with aromatic residues and arginine forming the key stabilising interactions. The total aromatic and arginine content of the two proteins must be appreciably different for distinct phases to form, and we show that this difference increases as the system is driven towards greater multiphasicity. Using the trends observed in the different interaction energies of this system, we demonstrate that we can also construct multilayered condensates with RNA preferentially concentrated in one phase. The 'rules' identified can thus enable the design of synthetic multiphasic condensates to facilitate further study of their organisation and function.

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multi-component mixtures of biomolecules (e.g. proteins and nucleic acids) when the different components present sufficient physicochemical diversity (e.g. in inter-molecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g. temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli, or progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of ageing. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically ageing condensates with non-conservative inter-molecular forces. Our nonequilibrium simulations of condensate ageing predict that single-component condensates that are initially homogeneous and liquid-like can transform into gel-core/liquid-shell or liquid-core/gel-shell multiphase condensates as they age, due to gradual and irreversible enhancement of inter-protein interactions. The type of multiphase architecture is determined by the ageing mechanism, the molecular organization of the gel and liquid phases, and the chemical make up of the protein. Notably, we predict that inter-protein disorder-to-order transitions within the prion-like domains of intracellular proteins could lead to the required non-conservative enhancement of inter-molecular interactions. Our study, therefore, predicts a potential mechanism Significance Statement Biomolecular condensates are highly diverse systems spanning not only homogeneous liquid droplets, but also gels, glasses, and even multiphase architectures that contain various coexisting liquid-like and/or gel-like inner phases. Multiphase architectures form when the different biomolecular components in a multi-component condensate establish sufficiently imbalanced inter-molecular forces to sustain different coexisting phases. While such a requirement seems, at first glance, impossible to fulfil for a condensate formed exclusively of chemically-identical proteins (i.e., single-component), our simulations predict conditions under which this may be possible. During condensate ageing, a sufficiently large imbalance in inter-molecular interactions can emerge intrinsically from the accumulation of protein structural transitions—driving even single-component condensates into nonequilibrium liquid-core/gel-shell or gel-core/liquid-shell multiphase architectures., Adiran Garaizar acknowledges funding from the EPRSC (EP/N509620)

Chromatin in eukaryotic cells is a negatively charged long polymer consisting of DNA, histones, and various associated proteins. With its highly charged and heterogeneous nature, chromatin structure varies greatly depending on various factors (e.g., chemical modifications and protein enrichment) and the surrounding environment (e.g., cations): From a 10-nm fiber, a folded 30-nm fiber, to chromatin condensates/droplets. Recent advanced imaging has observed that chromatin exhibits a dynamic liquid-like behavior and undergoes structural variations within the cell. Current computational modeling has made it possible to reconstruct the liquid-like chromatin in the cell by dealing with a number of nucleosomes on multi-scale levels, and has become a powerful technique to inspect the molecular mechanisms giving rise to the observed behavior, which imaging methods cannot do on their own. Based on new findings from both imaging and modeling studies, we discuss the dynamic aspect of chromatin in living cells and its functional relevance.