Search

Prototropic and solvatochromatic properties of fluorescein (FL) were employed to detect the presence of microenvironments in polyplexes consisting of polycationic polymer (POCP) and a fluorescein-conjugated RNA, the HIV-1 transactivation response element (TAR-FL). Results reveal new aspects of polyplex structure with respect to polyplex-bound RNA existing in the following local microenvironments: (a) RNA associated with the polyplex that experiences local pH changes in a manner dependent on POCP nitrogen to RNA phosphate ratio (N:P), (b) RNA experiencing relatively acidic local pH environment that remains constant in polyplexes formed after a charge-neutral ratio, and (c) RNA packed close enough to mediate fluorophore/fluorophore quenching. The magnitude of these changes observed as a function of POCP to nucleic acid N:P ratio is polymer dependent. Assessment of the different microenvironments can help elucidate the functional hierarchy of polyplex-bound oligonucleotides and additionally characterize POCPs based on the resulting local pH and solvent properties upon polyplex formation.

Phase separation of biological molecules, such as nucleic acids and proteins, has garnered widespread attention across many fields in recent years. For instance, liquid-liquid phase separation has been implicated not only in membraneless intracellular organization but also in many biochemical processes, including transcription, translation, and cellular signaling. Here, we present a historical background of biological phase separation and survey current work on nuclear organization and its connection to DNA phase separation from the perspective of DNA sequence, structure, and genomic context.

Complexation of oppositely charged macromolecules is essential in several biological phenomena. Recent reports have demonstrated the importance of DNA local flexibility in governing liquid–liquid phase separation (LLPS), a ubiquitous phenomenon thought to drive membraneless cellular organization. Inspired by this, we perform coarse-grained molecular dynamics simulations to study the role of chain flexibility in the complexation of short negatively charged polyelectrolytes with long positively charged polyelectrolytes. At low ionic strengths, spontaneous segregation of chains into a condensed (polymer-rich) phase and a supernatant (polymer-depleted) phase is observed. When both the polyanion and polycation are flexible (flexible–flexible complex), denser complexes with a higher degree of structural correlation form, with fewer free chains released into the supernatant, compared to the case when the polyanion is rigid (rigid–flexible complex), in agreement with the LLPS experiments. Free chains are in rapid...

Polydispersity and random sequences are ubiquitous features of polymers, and molecular dynamics simulations can help elucidate the impact of disorder in polymer systems. However, currently available packages for building polymer topologies do not enable the user to include randomness in a straightforward fashion. Here, we introduce Hoobas, a molecular builder package that easily handles polydispersity using a prototype-builder design pattern. This enables fast and easy building of systems comprised of thousands of distinct objects. It is written in the Python programming language, which ensures compatibility with a wide range of molecular dynamics packages and tools, as well as easy integration into most workflows.

Significance The interfacial physics of complex liquids under flow remains a long-standing problem in fluid mechanics that is important for fields ranging from lubrication to nanofluidics. Liquids containing small amounts of high-molecular weight polymers are found to flow through channels faster than expected, a phenomenon attributed to the formation of boundary depletion layers that relaxes the no-slip boundary condition and allows the bulk of the fluid to slip past the walls. This work provides the most direct measurement to date of the dimension and composition of depletion layers of a polymer solution under flow. We anticipate extending this approach to help understand fluid dynamics in different regimes, such as flow in nanoconfinement and turbulence., Complex liquids flow through channels faster than expected, an effect attributed to the formation of low-viscosity depletion layers at the boundaries. Characterization of depletion layer length scale, concentration, and dynamics has remained elusive due in large part to the lack of suitable real-space experimental techniques. The short length scales associated with depletion layers have traditionally prohibited direct imaging. By overcoming this limitation via adaptations of stimulated emission depletion (STED) microscopy, we directly measure the concentration profile of polymer solutions at a nonadsorbing wall under Poiseuille flow. Using this approach, we 1) confirm the theoretically predicted concentration profile governed by entropically driven depletion, 2) observe depletion layer narrowing at low to intermediate shear rates, and 3) report depletion layer composition that approaches pure solvent at unexpectedly low shear rates.

Phase separation of intracellular components has been recently realized as a mechanism by which cells achieve membraneless organization. Here, we study the associative liquid-liquid phase separation (LLPS) of DNA upon complexation with cationic polypeptides. Comparing the phase behavior of different single-stranded DNA as well as double-stranded DNA (dsDNA) sequences that differ in persistence lengths, we find that DNA local flexibility, not simply charge density, determines the LLPS. Furthermore, in a nucleotide- and DNA-dependent manner, free nucleotide triphosphates promote LLPS of polypeptide-dsDNA complexes that are otherwise prone to precipitation. Under these conditions, dsDNA undergoes a secondary phase separation forming liquid-crystalline subcompartments inside the droplets. These results point toward a role of local DNA flexibility, encoded in the sequence, in the regulation and selectivity of multicomponent LLPS in membraneless intracellular organization.

Bulk-level measurements of dynamics have suggested that phase-separated, protein-nucleic acid rich droplets can be viewed as simple liquids. In this report, we show that histone proteins spontaneously phase separate into liquid-like droplets in the presence of DNA. Using super-resolution fluorescence microscopy, we find that molecular transport in these droplets is non-Fickian (subdiffusive) at nanoscopic length scales. This observation cannot be explained by charge-charge interactions. Instead, our results strongly suggest that cation-π interactions drive the non-Fickian behavior. Given the ubiquity of cationic and aromatic moieties in protein-nucleic acid rich liquid-like phases observed in cells, we anticipate that non-Fickian diffusion is a general transport mechanism in such phases.

Liquid-liquid phase separation (LLPS) of proteins and nucleic acids has emerged as an important phenomenon in membraneless intracellular organization. We demonstrate that the linker histone H1 condenses into liquid-like droplets in the nuclei of HeLa cells. The droplets, observed during the interphase of the cell cycle, are colocalized with DNA-dense regions indicative of heterochromatin. In vitro, H1 readily undergoes LLPS with both DNA and nucleosomes of varying lengths but does not phase separate in the absence of DNA. The nucleosome core particle maintains its structural integrity inside the droplets, as demonstrated by FRET. Unexpectedly, H2A also forms droplets in the presence of DNA and nucleosomes in vitro, whereas the other core histones precipitate. The phase diagram of H1 with nucleosomes is invariant to the nucleosome length at physiological salt concentration, indicating that H1 is capable of partitioning large segments of DNA into liquid-like droplets. Of the proteins tested (H1, core histones, and the heterochromatin protein HP1α), this property is unique to H1. In addition, free nucleotides promote droplet formation of H1 nucleosome in a nucleotide-dependent manner, with droplet formation being most favorable with ATP. Although LLPS of HP1α is known to contribute to the organization of heterochromatin, our results indicate that H1 also plays a role. Based on our study, we propose that H1 and DNA act as scaffolds for phase-separated heterochromatin domains.

Cells achieve highly intricate internal organization via membrane-bound and membraneless organelles. Research over the past decade has implicated liquid–liquid phase separation, a phenomenon by which charged and often disordered biological macromolecules assemble into reversible liquid-like condensates, as the mechanism of formation of membraneless organelles in cells. During the same period, optical microscopy saw exciting advancements, including the super-resolution revolution, that were quickly adopted by researchers in the biological community. Today, there exists a vast library of techniques capable of providing unprecedented information regarding the formation, function, and dynamics of biomolecular condensates. In this review, we discuss a select number of modern optical microscopy methods that are particularly suited for studying biomolecular condensates both in vitro and in cells, as well as the associated technical challenges.

A combination of solution NMR, dynamic light scattering (DLS), and fluorescence quenching assays were employed to obtain insights into the dynamics and structural features of a polyplex system consisting of HIV-1 transactivation response element (TAR) and PEGylated generation 5 poly(amidoamine) dendrimer (G5-PEG). NMR chemical shift mapping and 13C spin relaxation based dynamics measurements depict the polyplex system as a highly dynamic assembly where the RNA, with its local structure and dynamics preserved, rapidly exchanges (< ms) between free and polyplex-bound forms, at least for the probed N:P ratios. Recovery of quenched fluorescence shows that RNA in the polyplex can be competitively exchanged over a wide range of amine to phosphate (N:P) charge ratios. The rapid exchange between free and bound forms may contribute to the mechanism by which RNA is released into the cell upon delivery while being protected by the polyplex environment from immediate degradation by nucleases.

Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

A kinetic model is proposed to predict the probability of dG•dT misincorporation across different polymerases, and provides mechanisms for sequence-dependent misincorporation. James Watson and Francis Crick are most remembered for their proposal regarding the B-form structure of DNA, but in their 1953 paper they also proposed that transient adoption of a tautomeric base pairing might lead to a spontaneous base change. In 2015, Hashim Al-Hashimi and team provided definitive evidence for the existence of these disfavoured tautomers. Now, Hashim Al-Hashimi and colleagues show the specific impact of these tautomers on DNA polymerase activity. They incorporate tautomerization and ionization data into a minimal kinetic model that is highly predictive of the misincorporation outcome by the polymerase. These results provide a greater understanding of the mutagenic process in cells. Tautomeric and anionic Watson–Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson–Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/U G•Tenol/Uenol; population less than 0.4%) and one anionic species (G•T–/U–; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10−2 to 10−3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT– dominant at pH 8.4 and above or for some mutagenic nucleotides.

Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.

The sensitization of p-GaP by adsorbed CdSe quantum dots has been observed. Nondegenerately doped, planar p-GaP(100) photoelectrodes consistently showed sub-band-gap (550 nm) photoresponsivity in an aqueous electrolyte containing Eu(3+/2+) when CdSe quantum dots (diameters ranging from 3.1 to 4.5 nm) were purposely adsorbed on the surface. Both time-resolved photoluminescence decays and steady-state photoelectrochemical responses supported sensitized hole injection from the CdSe quantum dots into p-GaP. The observation of hole injection in this system stands in contrast to sensitized electron injection seen in other metal oxide/quantum dot material combinations and therefore widens the possible designs for photoelectrochemical energy conversion systems that utilize quantum dots as light-harvesting components.

The equilibrium structure, stability, and electronic properties of the Al(13)X (X=H,Au,Li,Na,K,Rb,Cs) clusters have been studied using a combination of photoelectron spectroscopy experiment and density functional theory. All these clusters constitute 40 electron systems with 39 electrons contributed by the 13 Al atoms and 1 electron contributed by each of the X (X=H,Au,Li,Na,K,Rb,Cs) atom. A systematic study allows us to investigate whether all electrons contributed by the X atoms are alike and whether the structure, stability, and properties of all the magic clusters are similar. Furthermore, quantitative agreement between the calculated and the measured electron affinities and vertical detachment energies enable us to identify the ground state geometries of these clusters both in neutral and anionic configurations.

Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA The study of isolated protein complexes has greatly benefited from recent advances in mass spectrometry instrumentation and quantitative, isotope labeling techniques. The comprehensive characterization of protein complex components and quantification of their relative abundance relies heavily upon maximizing protein and peptide sequence information obtained from MS and tandem MS studies. Recent work has shown that using a metalloendopeptidase, Lys-N, for proteomic analysis of biological protein mixtures produces complementary protein sequence information compared with trypsin digestion alone. Here, we have investigated the suitability of Lys-N proteolysis for use with MALDI mass spectrometry to characterize the yeast Arp2 complex and E. coli PAP I protein interactions. Although Lys-N digestion resulted in an average decrease in protein sequence coverage of ∼30% compared with trypsin digestion, CID analysis of singly-charged Lys-N peptides yielded a more extensive b-ions series compared with complementary tryptic peptides. Taking advantage of this improved fragmentation pattern, we utilized differential 15N/14N guanidination of Lys-N peptides and MALDI-MS/MS analysis to relatively quantify the changes in PAP I associations due to deletion of sprE, previously shown to regulate PAP I-dependent polyadenylation. Overall, this Lys-N/guanidination integrative approach is applicable for functional proteomic studies utilizing MALDI mass spectrometry analysis, as it provides an effective and economical mean for relative quantification of proteins in conjunction with increased sensitivity of detection and fragmentation efficiency.