Abstract:Anionic macromolecular crowding promotes a very efficient compaction of chromatin fibers and self-assembly into micrometer-sized colloidal aggregates.
“…Depending on the characteristics of the DNA and the condensing agents in the solvent, condensates have been reported to be rather liquid-like or solid-like. Examples of experimentally observed DNA condensates are the toroidal or rod-like assemblies formed by DNA molecules in the presence of multivalent cations ( 6 , 9 ); the DNA gel particles obtained in concentrated protein solutions or in the presence of a cationic surfactant ( 10 ); the liquid crystalline condensates formed by mononucleosomes in crowded solutions ( 11 ); the condensates formed by 12-mer nucleosomal arrays in the presence of divalent cations and/or chromatin-associated proteins, which have been reported to behave rather liquid-like ( 12 , 13 ) or solid-like ( 14 ) depending on the solvent composition; and the rather solid-like aggregates formed by longer nucleosomal templates under conditions of anionic protein crowding ( 15 ). Figure 1 Predicted length dependence of DNA condensation.…”
Section: Introductionmentioning
confidence: 99%
“…The DNA molecules used to study condensates in vitro are typically much shorter than the megabase-sized chromosomes present in mammalian cells or the topologically associating domains with sizes of hundreds of kilobases into which chromosomes can be subdivided. Although toroidal assemblies and solid-like aggregates have also been observed for relatively long DNA molecules such as T4 phage DNA with a size of ∼170 kb ( 15 , 16 ), liquid-like condensates have mostly been observed for much shorter DNA molecules, e.g., ( 11 , 12 , 13 , 17 , 18 , 19 ). Although it has long been known that DNA length has an influence on the solubility and condensation behavior of DNA, e.g., ( 20 , 21 ), it is largely unclear how DNA length affects the material properties and in particular the fluidity of the resulting condensates in the presence of different condensing agents, making it difficult to compare previous in vitro experiments using differently sized DNA molecules to each other and to the situation in the cell.…”
Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage l-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1-10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.
“…Depending on the characteristics of the DNA and the condensing agents in the solvent, condensates have been reported to be rather liquid-like or solid-like. Examples of experimentally observed DNA condensates are the toroidal or rod-like assemblies formed by DNA molecules in the presence of multivalent cations ( 6 , 9 ); the DNA gel particles obtained in concentrated protein solutions or in the presence of a cationic surfactant ( 10 ); the liquid crystalline condensates formed by mononucleosomes in crowded solutions ( 11 ); the condensates formed by 12-mer nucleosomal arrays in the presence of divalent cations and/or chromatin-associated proteins, which have been reported to behave rather liquid-like ( 12 , 13 ) or solid-like ( 14 ) depending on the solvent composition; and the rather solid-like aggregates formed by longer nucleosomal templates under conditions of anionic protein crowding ( 15 ). Figure 1 Predicted length dependence of DNA condensation.…”
Section: Introductionmentioning
confidence: 99%
“…The DNA molecules used to study condensates in vitro are typically much shorter than the megabase-sized chromosomes present in mammalian cells or the topologically associating domains with sizes of hundreds of kilobases into which chromosomes can be subdivided. Although toroidal assemblies and solid-like aggregates have also been observed for relatively long DNA molecules such as T4 phage DNA with a size of ∼170 kb ( 15 , 16 ), liquid-like condensates have mostly been observed for much shorter DNA molecules, e.g., ( 11 , 12 , 13 , 17 , 18 , 19 ). Although it has long been known that DNA length has an influence on the solubility and condensation behavior of DNA, e.g., ( 20 , 21 ), it is largely unclear how DNA length affects the material properties and in particular the fluidity of the resulting condensates in the presence of different condensing agents, making it difficult to compare previous in vitro experiments using differently sized DNA molecules to each other and to the situation in the cell.…”
Living organisms typically store their genomic DNA in a condensed form. Mechanistically, DNA condensation can be driven by macromolecular crowding, multivalent cations, or positively charged proteins. At low DNA concentration, condensation triggers the conformational change of individual DNA molecules into a compacted state, with distinct morphologies. Above a critical DNA concentration, condensation goes along with phase separation into a DNA-dilute and a DNA-dense phase. The latter DNA-dense phase can have different material properties and has been reported to be rather liquid-like or solid-like depending on the characteristics of the DNA and the solvent composition. Here, we systematically assess the influence of DNA length on the properties of the resulting condensates. We show that short DNA molecules with sizes below 1 kb can form dynamic liquid-like assemblies when condensation is triggered by polyethylene glycol and magnesium ions, binding of linker histone H1, or nucleosome reconstitution in combination with linker histone H1. With increasing DNA length, molecules preferentially condense into less dynamic more solid-like assemblies, with phage l-DNA with 48.5 kb forming mostly solid-like assemblies under the conditions assessed here. The transition from liquid-like to solid-like condensates appears to be gradual, with DNA molecules of roughly 1-10 kb forming condensates with intermediate properties. Titration experiments with linker histone H1 suggest that the fluidity of condensates depends on the net number of attractive interactions established by each DNA molecule. We conclude that DNA molecules that are much shorter than a typical human gene are able to undergo liquid-liquid phase separation, whereas longer DNA molecules phase separate by default into rather solid-like condensates. We speculate that the local distribution of condensing factors can modulate the effective length of chromosomal domains in the cell. We anticipate that the link between DNA length and fluidity established here will improve our understanding of biomolecular condensates involving DNA.
“…Although many studies have addressed the structure and dynamics of smaller segments of chromatin (12,13), only a few have focused on larger chromatins that provide a good model system for TADs. Here, we will use reconstituted chromatin by complexation of over-expressed, refolded, and purified histone octamer on bacteriophage T4-DNA (165.6 kbp) (14)(15)(16). This system can serve as a model of (smaller) TADs because it approaches their molecular weight, there are no preferential histone binding sites, and the flexibility in nucleosome position is generally conserved in vivo human chromatin (17).…”
“…This loss of target specificity with increased CW800 labeling has been reported before by Choi and co-workers who observed higher background fluorescence with secondary antibodies labeled with CW800 (DOL 2.5) compared to the versions with a lower DOL of 1.2 . We speculate that the undesired nuclear accumulation of 2’Ab-CW800 (DOL 4.4) is due to the strong electrostatic attraction of the anionic dye-labeled conjugate to cationic histones in the nucleus . Because 2’Ab-CW800 (DOL 1.8) exhibited the cleanest targeting of primary anti-tubulin with a minimal nonspecific signal, it was chosen as a near-optimal 2’Ab-CW800 conjugate for comparison studies.…”
A broad array of imaging and diagnostic
technologies employs fluorophore-labeled
antibodies for biomarker visualization,
an experimental technique known as immunofluorescence. Significant
performance advantages, such as higher signal-to-noise ratio, are
gained if the appended fluorophore emits near-infrared (NIR) light
with a wavelength >700 nm. However, the currently available NIR
fluorophore
antibody conjugates are known to exhibit significant limitations,
including low chemical stability and photostability, weakened target
specificity, and low fluorescence brightness. These fluorophore limitations
are resolved by employing a NIR heptamethine cyanine dye named s775z
whose chemical structure is very stable, charge-balanced, and sterically
shielded. Using indirect immunofluorescence for imaging and visualization,
a secondary IgG antibody labeled with s775z outperformed IgG analogues
labeled with the commercially available NIR fluorophores, IRDye 800CW
and DyLight800. Comparison experiments include three common techniques:
immunocytochemistry, immunohistochemistry, and western blotting. Specifically,
the secondary IgG labeled with s775z was 3–8 times brighter,
3–6 times more photostable, and still retained excellent target
specificity when the degree of antibody labeling was high. The results
demonstrate that antibodies labeled with s775z can emit total photon
counts that are 1–2 orders of magnitude higher than those currently
possible, and thus enable unsurpassed performance for NIR fluorescence
imaging and diagnostics. They are especially well suited for analytical
applications that require sensitive NIR fluorescence detection or
use modern photon-intense methods that require high photostability.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
