Quantification of the concentration in a droplet formed by liquid–liquid phase separation of G-quadruplex-forming RNA

Yokosawa, Kohei; Tsuruta, Mitsuki; Kajimoto, Shinji; Sugimoto, Naoki; Miyoshi, Daisuke; Nakabayashi, Takakazu

doi:10.1016/j.cplett.2023.140634

Cited by 6 publications

(4 citation statements)

References 31 publications

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“…3b ) indicates nearly complete recruitment of clp and polyU molecules to droplets, which brings the paramagnetic TOAC-labeled clp molecules close to diamagnetic clp and polyU molecules and consequently causes a paramagnetic relaxation enhancement (PRE)-induced broadening of most of their NMR signals. A similar behavior has been recently reported for the RNA-induced phase separation of an RGG-rich peptide, for which Raman microscopy data demonstrated the full peptide recruitment into formed droplets 39 . In 2D 1 H, 1 H TOCSY spectrum of the mixture of dia/paramagnetic clp and polyU, the HN-HA correlation peaks from the C-terminal residues V423 and S424, and to a lower extent, several other residues especially close to the C-terminus of peptide, partially survived, indicating their relatively high residual reorientational mobility inside the formed droplets (Fig.…”

Section: Resultssupporting

confidence: 84%

“…Indeed, it paves the way to exploit this and other 2D-ESEEM techniques within the context of phase separation especially when the condensed phase is highly crowded, widening the range of putative hyperfine couplings with the paramagnetic probe. Notably, a more than 1000-fold increase in protein concentration inside droplets has been recently reported for an RGG-rich peptide undergoing RNA-induced phase separation 39 , and high protein mass concentration inside droplets in the range of 100–700 mg/mL concentrations are reported for tau protein, FUS-LC, an RGG-rich peptide and ATXN3 39 , 53 – 55 .…”

Section: Discussionmentioning

confidence: 94%

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Large dynamics of a phase separating arginine-glycine-rich domain revealed via nuclear and electron spins

Sicoli,

Sieme,

Overkamp

et al. 2024

Nat Commun

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Liquid-liquid phase separation is the key process underlying formation of membrane-less compartments in cells. A highly dynamic cellular body with rapid component exchange is Cajal body (CB), which supports the extensive compositional dynamics of the RNA splicing machinery, spliceosome. Here, we select an arginine-glycine (RG)-rich segment of coilin, the major component of CB, establish its RNA-induced phase separation, and through combined use of nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) probes, interrogate its dynamics within the crowded interior of formed droplets. Taking advantage of glycine-based singlet-states, we show that glycines retain a large level of sub-nanoseconds dynamics inside the coilin droplets. Furthermore, the continuous-wave (CW) and electron-electron dipolar (PELDOR) and electron-nucleus hyperfine coupling EPR data (HYSCORE) support the RNA-induced formation of dynamic coilin droplets with high coilin peptide concentrations. The combined NMR and EPR data reveal the high dynamics of the RG-rich coilin within droplets and suggest its potential role in the large dynamics of CBs.

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Section: Resultssupporting

confidence: 84%

Section: Discussionmentioning

confidence: 94%

Large dynamics of a phase separating arginine-glycine-rich domain revealed via nuclear and electron spins

Sicoli,

Sieme,

Overkamp

et al. 2024

Nat Commun

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“…As mentioned in , we have reported the determination of the protein concentration in an LLPS droplet in buffer solution using the Raman band of water outside the droplets as an intensity standard. − Quantification using the water Raman band is based on the fact that the concentration of water in liquid water is extremely high and constant. In this study, we applied this method to quantify the intracellular components.…”

Section: Resultsmentioning

confidence: 99%

“…In previous studies, we propose that the Raman band of water can be used as an intensity standard for determining the concentration of the target molecule using Raman microscopy. − We demonstrated the concentration quantification using liquid droplets of the protein formed by liquid–liquid phase separation (LLPS) in a buffer solution. − Taking advantage of the fact that the concentration of water molecules in water outside the droplet is constant regardless of the sample, we normalized the Raman band of the protein in a droplet by the O–H stretching Raman band of water outside the droplet and determined the protein concentration within a single droplet in a label-free manner. We also applied this method to cells and succeeded in quantifying the concentration of nucleic acids in a living cell by normalizing their Raman intensities with the O–H stretching band of water outside the cells as an intensity standard, utilizing the constant concentration of extracellular water …”

Section: Introductionmentioning

confidence: 99%

Establishment of a Method for the Introduction of Poorly Water-Soluble Drugs in Cells and Evaluation of Intracellular Concentration Distribution Using Resonance Raman Imaging

Koga,

Kajimoto,

Yoshizaki

et al. 2024

J. Phys. Chem. B

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Label-free measurement is essential to understand the metabolism of drug molecules introduced into cells. Raman imaging is a powerful method to investigate intracellular drug molecules because it provides in situ label-free observation of introduced molecules. In this study, we propose that Raman imaging can be used not only to observe the intracellular distribution of drug molecules but also to quantitatively visualize the concentration distribution reflecting each organelle in a single living cell using the Raman band of extracellular water as an intensity standard. We dissolved poorly water-soluble all-trans-retinoic acid (ATRA) in water using a cytocompatible amphiphilic phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate] (PMB) as a solubilizing reagent, introduced it into cells, and obtained the intracellular concentration distribution of ATRA. ATRA was concentrated in the cells and mainly localized to mitochondria and lipid droplets, interacting strongly with mitochondria and weakly with lipid droplets. Poorly water-soluble β-carotene was also introduced into cells using PMB but was not concentrated intracellularly, indicating that βcarotene does not interact specifically with intracellular molecules. We established a protocol for the solubilization and intracellular uptake of poorly water-soluble molecules using PMB and obtaining their concentration distribution using Raman microscopy.

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Factors Affecting Liquid‐Liquid Phase Separation of RGG Peptides with DNA G‐Quadruplex

Shil,

Tsuruta,

Kawauchi

et al. 2024

ChemMedChem

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Liquid‐liquid phase separation (LLPS), mediated by G‐quadruplexes (G4 s) and intrinsically disordered proteins, particularly those containing RGG domains, plays a critical role in cellular processes and diseases. However, the molecular mechanism and the role of individual amino acid residues of the protein in LLPS with G4 (G4‐LLPS) are still unknown. Here, we systematically designed peptides and investigated the roles of arginine residues in G4‐LLPS. It was found that the FMRP‐derived RGG peptide induced LLPS with G4‐forming Myc‐DNA, whereas a point‐mutated peptide, in which all arginine residues were replaced with lysine, was unable to undergo LLPS, indicating the importance of arginine residues. Moreover, systematically truncated peptides showed that at least five positive net charges of peptide are required to induce G4‐LLPS. Furthermore, quantitative investigation demonstrated that the higher binding affinity of peptides with G4 led to a higher LLPS ability, whereas threshold of the binding affinity for undergoing LLPS was identified. These insights elucidate the pivotal role of arginine in G4‐LLPS and the specific requirement for multiple arginine residues, contributing to a deeper understanding of the complex interplay between intrinsically disordered proteins and nucleic acids.

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Quantification of the concentration in a droplet formed by liquid–liquid phase separation of G-quadruplex-forming RNA

Cited by 6 publications

References 31 publications

Large dynamics of a phase separating arginine-glycine-rich domain revealed via nuclear and electron spins

Large dynamics of a phase separating arginine-glycine-rich domain revealed via nuclear and electron spins

Establishment of a Method for the Introduction of Poorly Water-Soluble Drugs in Cells and Evaluation of Intracellular Concentration Distribution Using Resonance Raman Imaging

Factors Affecting Liquid‐Liquid Phase Separation of RGG Peptides with DNA G‐Quadruplex

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