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Near-identical macromolecules spontaneously partition into concentric circles

Nature volume 636, pages 92–99 (2024)Cite this article

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Abstract

Although separation is entropically unfavourable, it is often essential for our life1,2. The separation of very similar macromolecules such as deoxyribonucleic acids (DNAs) and their single nucleotide variants is difficult but holds great advantage for the progress of life science3. Here we report that a particular liquid–liquid phase separation (LLPS) at a solid–liquid interface led to the partitioning of DNAs with nearly identical structures. We found this intriguing phenomenon when we did drop-casting onto a glass plate an aqueous ammonium sulfate dispersion of phase-separated droplets comprising a homogeneous mixture of poly(ethylene glycol) (PEG) samples with different termini. Even when the molecular weights of their PEG parts were identical to each other, terminally different PEGs spread competitively at the solid–liquid interface and partitioned into micrometre-scale concentric circles. We found that this competitive spreading was induced by an ammonium sulfate layer spontaneously formed on the glass surface. We successfully extended the above mechanism to partitioning a mixture of nearly identical DNAs into concentric circles followed by their selective extraction using the salting-in effect. We could isolate a human cancer-causing single nucleotide variant in 97% purity from its 1:1 mixture with the original DNA.

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Fig. 1: Partitioning of nearly identical macromolecules into concentric circles.
Fig. 2: Mechanistic studies of concentric partitioning.
Fig. 3: Selective extraction of concentrically partitioned DNAs by the salting-in effect.
Fig. 4: Post-hybridization to visualize concentrically partitioned mixtures of DNAs.

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References

  1. Seader, J. D., Henley, E. J. & Roper, D. K. Separation Process Principles (Wiley, 2011).

  2. Baker, R. W. Membrane Technology and Applications (Wiley, 2023).

  3. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002).

    ADS  CAS  PubMed  Google Scholar 

  4. Welton, T. & Reichardt, C. Solvents and Solvent Effects in Organic Chemistry (Wiley, 2011).

  5. Grover, P. K. & Ryall, R. L. Critical appraisal of salting-out and its implications for chemical and biological sciences. Chem. Rev. 105, 1–10 (2005).

    CAS  PubMed  Google Scholar 

  6. Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Arakawa, T. & Timasheff, S. N. Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry 23, 5912–5923 (1984).

    CAS  PubMed  Google Scholar 

  8. Langmuir, I. The role of attractive and repulsive forces in the formation of tactoids, thixotropic gels, protein crystals and coacervates. J. Chem. Phys. 6, 873–896 (1938).

    ADS  CAS  Google Scholar 

  9. Zhang, F. et al. Reentrant condensation, liquid–liquid phase separation and crystallization in protein solutions induced by multivalent metal ions. Pure Appl. Chem. 86, 191–202 (2014).

    CAS  Google Scholar 

  10. Luisi, P. The Emergence of Life (Cambridge Univ. Press, 2006).

  11. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Gouveia, B. et al. Capillary forces generated by biomolecular condensates. Nature 609, 255–264 (2022).

    ADS  CAS  PubMed  Google Scholar 

  13. Zarzar, L. D. et al. Dynamically reconfigurable complex emulsions via tunable interfacial tensions. Nature 518, 520–524 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Nagelberg, S. et al. Reconfigurable and responsive droplet-based compound micro-lenses. Nat. Commun. 8, 14673 (2017).

    ADS  PubMed  PubMed Central  Google Scholar 

  15. Goodling, A. E. et al. Colouration by total internal reflection and interference at microscale concave interfaces. Nature 566, 523–527 (2019).

    ADS  CAS  PubMed  Google Scholar 

  16. Concellón, A., Fong, D. & Swager, T. M. Complex liquid crystal emulsions for biosensing. J. Am. Chem. Soc. 143, 9177–9182 (2021).

    PubMed  Google Scholar 

  17. Cacace, M. G., Landau, E. M. & Ramsden, J. J. The Hofmeister series: salt and solvent effects on interfacial phenomena. Q. Rev. Biophys. 30, 241–277 (1997).

    CAS  PubMed  Google Scholar 

  18. Duong-Ly, K. C. & Gabelli, S. B. Salting out of proteins using ammonium sulfate precipitation. Methods Enzymol. 541, 85–94 (2014).

    CAS  PubMed  Google Scholar 

  19. Bailey, F. E. Jr. & Callard, R. W. Some properties of poly(ethylene oxide)1 in aqueous solution. J. Appl. Polym. Sci. 1, 56–62 (1959).

    CAS  Google Scholar 

  20. Kim, C. W. & Rha, C. Phase separation of polyethylene glycol/salt aqueous two-phase systems. Phys. Chem. Liquids 38, 181–191 (2000).

    CAS  Google Scholar 

  21. Chao, Y. & Shum, H. C. Emerging aqueous two-phase systems: from fundamentals of interfaces to biomedical applications. Chem. Soc. Rev. 49, 114–142 (2020).

    CAS  PubMed  Google Scholar 

  22. Watanabe, C. et al. Cell-sized confinement initiates phase separation of polymer blends and promotes fractionation upon competitive membrane wetting. ACS Mater. Lett. 4, 1742–1748 (2022).

    CAS  Google Scholar 

  23. Mangiarotti, A., Chen, N., Zhao, Z., Lipowsky, R. & Dimova, R. Wetting and complex remodeling of membranes by biomolecular condensates. Nat. Commun. 14, 2809 (2023).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mori, S. & Barth, H. G. Size Exclusion Chromatography (Springer, 2011).

  25. Snyder, L. R., Kirkland, J. J. & Dolan, J. W. Introduction to Modern Liquid Chromatography (Wiley, 2010).

  26. Deegan, R. D. et al. Capillary flow as the cause of ring stains from dried liquid drops. Nature 389, 827–829 (1997).

    ADS  CAS  Google Scholar 

  27. Yunker, P. J., Still, T., Lohr, M. A. & Yodh, A. G. Suppression of the coffee-ring effect by shape-dependent capillary interactions. Nature 476, 308–311 (2011).

    ADS  CAS  PubMed  Google Scholar 

  28. Wong, T.-S., Chen, T.-H., Shen, X. & Ho, C.-M. Nanochromatography driven by the coffee ring effect. Anal. Chem. 83, 1871–1873 (2011).

    CAS  PubMed  Google Scholar 

  29. Tanner, L. H. The spreading of silicone oil drops on horizontal surfaces. J. Phys. D Appl. Phys. 12, 1473–1484 (1979).

    ADS  CAS  Google Scholar 

  30. Bonn, D., Eggers, J., Indekeu, J., Meunier, J. & Rolley, E. Wetting and spreading. Rev. Mod. Phys. 81, 739–805 (2009).

    ADS  CAS  Google Scholar 

  31. Hayes, R., Warr, G. G. & Atkin, R. Structure and nanostructure in ionic liquids. Chem. Rev. 115, 6357–6426 (2015).

    CAS  PubMed  Google Scholar 

  32. Mezger, M. et al. Molecular layering of fluorinated ionic liquids at a charged sapphire (0001) surface. Science 322, 424–428 (2008).

    ADS  CAS  PubMed  Google Scholar 

  33. Sauerbrey, G. The use of quartz oscillators for weighing thin layers and for microweighing. Z. Fur. Phys. 155, 206–222 (1959).

    ADS  CAS  Google Scholar 

  34. Pappu, R. V., Cohen, S. R., Dar, F., Farag, M. & Kar, M. Phase transitions of associative biomacromolecules. Chem. Rev. 123, 8945–8987 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Qian, D. et al. Tie-line analysis reveals interactions driving heteromolecular condensate formation. Phys. Rev. 12, 041038 (2022).

    CAS  Google Scholar 

  36. Zhang, W. et al. Liquid–liquid equilibrium of aqueous two-phase systems containing poly(ethylene glycol) of different molecular weights and several ammonium salts at 298.15 K. Thermochim. Acta 560, 47–54 (2013).

    CAS  Google Scholar 

  37. Wysoczanska, K. & Macedo, E. A. Influence of the molecular weight of PEG on the polymer/salt phase diagrams of aqueous two-phase systems. J. Chem. Eng. Data 61, 4229–4235 (2016).

    CAS  Google Scholar 

  38. Zhao, X. et al. Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth. Cell 186, 5517–5535 (2023).

    CAS  PubMed  Google Scholar 

  39. Entelis, S. G., Evreinov, V. V. & Gorshkov, A. V. Functionality and molecular weight distribution of telechelic polymers. Adv. Polym. Sci. 76, 129–175 (1987).

    Google Scholar 

  40. Gorbunov, A. & Trathnigg, B. Theory of liquid chromatography of mono- and difunctional macromolecules: I. Studies in the critical interaction mode. J. Chromatogr. A 955, 9–17 (2002).

    CAS  PubMed  Google Scholar 

  41. Le Ouay, B. et al. Selective sorting of polymers with different terminal groups using metal-organic frameworks. Nat. Commun. 9, 3635 (2018).

    ADS  PubMed  PubMed Central  Google Scholar 

  42. Peng, S. et al. Efficient separation of nucleic acids with different secondary structures by metal–organic frameworks. J. Am. Chem. Soc. 142, 5049–5059 (2020).

    CAS  PubMed  Google Scholar 

  43. Schneider, C., Rasband, W. & Eliceiri, K. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Hau, W. L. W., Trau, D. W., Sucher, N. J., Wong, M. & Zohar, Y. Surface-chemistry technology for microfluidics. J. Micromech. Microeng. 13, 272–278 (2003).

    ADS  CAS  Google Scholar 

  45. Chakraborty, A. & Sen, K. Impact of pH and temperature on phase diagrams of different aqueous biphasic systems. J. Chromatogr. A 1433, 41–55 (2016).

    CAS  PubMed  Google Scholar 

  46. Miller, W. L. & McPherson, R. H. The behavior of colloidal suspensions with immiscible solvents. J. Phys. Chem. 12, 706–716 (1908).

    Google Scholar 

  47. Williamson, J. C. Liquid–liquid demonstrations: phase equilibria and the lever rule. J. Chem. Educ. 98, 2356–2363 (2021).

    CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Fujihara, C.-H. Chen, B. Shen (The University of Tsukuba), H. Kitahata (Chiba University), J, Shiomi (The University of Tokyo) and E. Nakamura (The University of Tokyo) for their discussions on this project. We thank K. Adachi (RIKEN) and D. Hashizume (RIKEN) for helping with the X-ray diffraction analysis. T.A. acknowledges the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Specially Promoted Research (23H05408) on ‘Solid-State Materials Science of Supramolecular Polymers and Their Applications’. T.A. thanks Kao Corporation for their financial support. H.G. acknowledges the Materials Education program for the future leaders in Research, Industry, and Technology (MERIT) and the JSPS Fellowship for Young Scientists. M.Y. acknowledges the JSPS Grant-in-Aid for Scientific Research (21H05871, 22H01188 and 24H02287) and Japan Science and Technology (JST) Agency Program FOREST (JPMJFR213Y). T.S. acknowledges the Exploratory Research for Advanced Technology (ERATO, JPMJER2002) programme from JST.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan

    Hao Gong, Yuriko Sakaguchi, Tsutomu Suzuki & Takuzo Aida

  2. Komaba Institute for Science, The University of Tokyo, Tokyo, Japan

    Miho Yanagisawa

  3. Department of Basic Science, The University of Tokyo, Tokyo, Japan

    Miho Yanagisawa

  4. RIKEN Center for Emergent Matter Science, Wakō, Japan

    Takuzo Aida

Authors
  1. Hao Gong
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  2. Yuriko Sakaguchi
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Contributions

H.G. and T.A. conceived the project, designed the experiments and wrote the paper. H.G., T.A. and M.Y. constructed the theoretical part. H.G. performed most of the experiments. H.G. and M.Y. conducted the density measurements. Y.S. and T.S. performed quantitative DNA analysis. All authors discussed the results and commented on the paper.

Corresponding authors

Correspondence to Hao Gong or Takuzo Aida.

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Competing interests

A patent covering the partitioning–extraction process described in this work (number JP2024-188368) has been filed by the University of Tokyo, Japan, listing T.A., H.G., T.S., Y.S. and M.Y. as inventors. The authors declare no other interests.

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Extended data figures and tables

Extended Data Fig. 1 Effects of different salts on concentric partitioning.

a–d, Confocal laser scanning microscopy images for λext = 552 nm (RhB) of an aqueous 3:7 mixture of MePEG5KRhB and HPEG35KOH under the conditions for concentric partitioning using different salting-out reagents: a, (NH4)2SO4, b, (NH4)3PO4, c, Cs2SO4, d, NaCl.

Extended Data Fig. 2 Effects of initial mixing ratio and [(NH4)2SO4] on concentric partitioning.

a, Optical microscopic images of a concentrically partitioned 23:77 mixture of MePEG5KNBD and MePEG5KNH2: (i) Confocal laser scanning microscopy (CLSM) image for λext = 488 nm (NBD), (ii) bright-field image, (iii) phase contrast microscopy image, (iv) differential interference contrast microscopy image, and (v) three-dimensionally reconstructed CLSM image. Scale bars, 10 µm. Note: The mixing ratio was confirmed by 1H NMR, where the unreacted residue MePEG5KNH2 for the synthesis of MePEG5KNBD was included as shown in b. b, CLSM images for λext = 488 nm (NBD) (upper) of MePEG5KNH2 and MePEG5KNBD with different mixing ratios under the conditions for the concentric partitioning, together with their magnified CLSM images (middle) and normalized fluorescence intensity distributions along the pink-colored broken lines (lower). Ratios (weight): (i) 90/10, (ii) 75/25, (iii) 50/50, (iv) 25/75, and (v) 0/100. White scale bars, 10 µm. Right inset: three-dimensionally reconstructed CLSM image of the sample in (v). Yellow scale bar, 2 µm. c, CLSM images for λext = 488 nm (NBD) of a 1:1 mixture of MePEG5KNBD and MePEG5KNH2 under the conditions for concentric partitioning using different concentrations of (NH4)2SO4 (upper bar), together with their normalized fluorescence intensity distributions along the pink broken lines. Scale bars, 10 µm.

Extended Data Fig. 3 Time-of-flight secondary ion mass spectrometry mapping of a concentrically partitioned PEG mixture.

(i)–(ii) Confocal laser scanning microscopy (CLSM) images for λext = 552 nm (RhB) and (iii)–(vi) time-of-flight secondary ion mass spectrometry (TOF-SIMS) maps of a concentrically partitioned 3:7 mixture of MePEG5KRhB and HPEG35KOH. (iii)–(vi) TOF-SIMS maps based on overlay, C2H5O+ (m/z 45.03), NH4+ (m/z 18.06), and RhB– (m/z 397.21). Scale bars, 20 µm.

Extended Data Fig. 4 Fluorescence spectra of solution samples.

a–b, Fluorescence spectra (λext = 488 nm) of MePEG5KNBD (green curve, a), MePEG5KRhB (red curve, a), and a 3:1 mixture of MePEG5KNBD and MePEG5KRhB (black curve, b) in water. Combined spectrum (black broken curve, a) resulting from summing the spectral data of individual PEG. c–d, Fluorescence spectra (λext = 488 nm) of MePEG5KNBD (green curve, c), MePEG5KRhB (red curve, c), and a 3:1 mixture of MePEG5KNBD and MePEG5KRhB (blue curve, d) in aqueous (NH4)2SO4 solution (26.4 wt%). Combined spectrum (blue broken curve, c) resulting from summing the spectral data of individual PEG.

Extended Data Fig. 5 Fluorescence spectra of concentrically partitioned samples.

Normalized fluorescence spectra (λext = 488 nm) (magenta plots) of a concentrically partitioned 3:1 mixture of MePEG5KNBD and MePEG5KRhB on a glass plate. Extracted normalized fluorescence spectra from the periphery (green plots) and core (red plots) domain. Inset: confocal laser scanning microscopy (CLSM) image of the concentrically partitioned mixture. Scale bars, 30 µm.

Extended Data Fig. 6 X-ray diffraction analysis of the spontaneously formed (NH4)2SO4 layer.

a, Out-of-plane X-ray diffraction (XRD) analysis of glass plates at ambient conditions covered by 500-µL aqueous (NH4)2SO4 solution (52.8 wt%, black curve) and 500-µL milli-Q water (pink curve). b, XRD analysis of 20-µL aqueous (NH4)2SO4 solutions (52.8 wt%; black curve, 26.4 wt%; grey curve) and 20-µL milli-Q water (pink curve) in glassy capillary tubes (φ = 0.8 mm).

Extended Data Fig. 7 Selective extraction of a concentrically partitioned PEG mixture.

Time-dependent confocal laser scanning microscopy (CLSM) images for λext = 488 nm (NBD); λext = 552 nm (RhB) of a concentrically partitioned 1:1 mixture of terminally different PEGs (MePEG5KNBD and MePEG5KRhB) upon selective extraction. The sample was initially extracted with a flow of milli-Q water for 30 s, followed by a water–methanol mixture (80/20, v/v) for an additional 30 s, and finally a water–methanol mixture (50/50, v/v) for a minute. Residual fluorescent intensity ratios of PEGs are presented at the top left of the images. Scale bars, 12 µm.

Extended Data Fig. 8 Extraction of a random PEGs mixture.

Time-dependent confocal laser scanning microscopy (CLSM) images for λext = 488 nm (NBD); λext = 552 nm (RhB) of a random 1:1 mixture of terminally different PEGs (MePEG5KNBD and MePEG5KRhB) upon selective extraction. The sample was initially extracted with a flow of milli-Q water for 30 s, followed by a water–methanol mixture (80/20 v/v) for an additional 30 s, and finally a water–methanol mixture (50/50 v/v) for a minute. Residual fluorescent intensity ratios of PEGs are presented at the top left of the images. Scale bars, 50 µm.

Extended Data Fig. 9 Concentric partitioning of terminally different RNAs.

Confocal laser scanning microscopy image for λext = 488 nm (FAM); λext = 638 nm (Cy5) of a concentrically partitioned 1:1 mixture of terminally different RNAs, together with a magnified CLSM image (middle upper) and its normalized fluorescence intensity distributions (middle lower) along a line across the image center. Yellow scale bars, 5 µm. White scale bars, 2 µm.

Extended Data Fig. 10 Phase behaviours of nucleic acids.

Critical points in binodal curves of terminally and sequentially different DNAs and RNAs (10 µwt%) in an aqueous solution of (NH4)2SO4 at 20 °C analyzed using a node determination method via dynamic light scattering (DLS). The error bars represent the standard deviations of three samples.

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Gong, H., Sakaguchi, Y., Suzuki, T. et al. Near-identical macromolecules spontaneously partition into concentric circles. Nature 636, 92–99 (2024). https://doi.org/10.1038/s41586-024-08203-4

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