Phase separation of DNA: From past to present.

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.

Liquid-Liquid Phase Separation of Histone Proteins in Cells: Role in Chromatin Organization.

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.

Depletion layer dynamics of polyelectrolyte solutions under Poiseuille flow.

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.

DNA Local-Flexibility-Dependent Assembly of Phase-Separated Liquid Droplets.

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.

Non-Fickian Molecular Transport in Protein-DNA Droplets.

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.

Dynamic basis for dG•dT misincorporation via tautomerization and ionization.

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 (G•T/UG•T/U; population less than 0.4%) and one anionic species (G•T/U; population around 0.

Three RNA Microenvironments Detected in Fluxional Gene Delivery Polyplex Nanoassemblies.

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.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

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.

Rapid Exchange Between Free and Bound States in RNA-Dendrimer Polyplexes: Implications on the Mechanism of Delivery and Release.

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 (13)C 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 ( View Article and Find Full Text PDF

Polyplex-induced cytosolic nuclease activation leads to differential transgene expression.

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.

Sensitization of p-GaP with CdSe quantum dots: light-stimulated hole injection.

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.

Electronic structure and properties of isoelectronic magic clusters: Al13X (X=H,Au,Li,Na,K,Rb,Cs).

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.

Integrating Lys-N proteolysis and N-terminal guanidination for improved fragmentation and relative quantification of singly-charged ions.

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.