Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

Abstract

Background It is becoming clearer that living cells use water/water (w/w) phase separation to form membraneless organelles that exhibit various important biological functions. Currently, it is believed that the specific localization of biomacromolecules, including DNA, RNA and proteins in w/w microdroplets is closely related to their bio-activity. Despite the importance of this possible role of micro segregation, our understanding of the underlying physico-chemical mechanism is still unrefined. Further research to unveil the underlying mechanism of the localization of macromolecules in relation to their steric conformation in w/w microdroplets is needed. Principal findings Single-DNA observation of genome-size DNA (T4 GT7 bacteriophage DNA; 166kbp) by fluorescence microscopy revealed that DNAs are spontaneously incorporated into w/w microdroplets generated in a binary aqueous polymer solution with polyethylene glycol (PEG) and dextran (DEX). Interestingly, DNAs with elongated coil and shrunken conformations exhibit Brownian fluctuation inside the droplet. On the other hand, tightly packed compact globules, as well as assemblies of multiple condensed DNAs, tend to be located near the interface in the droplet. Conclusion and significance The specific localization of DNA molecules depending on their higher-order structure occurs in w/w microdroplet phase-separation solution under a binary aqueous polymer solution. Such an aqueous solution with polymers mimics the crowded conditions in living cells, where aqueous macromolecules exist at a level of 30–40 weight %. The specific positioning of DNA depending on its higher-order structure in w/w microdroplets is expected to provide novel insights into the mechanism and function of membraneless organelles and micro-segregated particles in living cells.

DNA compaction enhances the sensitivity of fluorescence-based nucleic acid assays: a game changer in point of care sensors?

“…39 As a consequence, the abrupt contraction of the DNA coil, known as, “coil–globule transition” takes place at a critical PEG concentration. 40…”

Cell-Size Space Regulates the Behavior of Confined Polymers: From Nano- and Micromaterials Science to Biology

“…This spindle size scaling by the space size can be easily reproduced by confining the cell extract in droplets of different space sizes (Figure d). , Studies suggest that the scaling mechanism is explained by finite elementality and the large surface area–volume ratio . In vitro experiments with genome-size DNA showed that its membrane interaction is associated with TX–TL dynamics and the higher-ordered structure of DNA. , …”

“…99 In vitro experiments with genome-size DNA showed that its membrane interaction is associated with TX−TL dynamics and the higher-ordered structure of DNA. 100,101 Cell-sized space also regulates higher-order structures other than genomic DNA. The spatial limitation plays a significant role in the macroscopic structure formation and function of molecules such as actin that polymerize like filaments and generate force for cell deformation.…”

Water‐in‐Water Droplets Selectively Uptake Self‐Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology

“…[15][16][17][18][19] We also observed that genomic DNA and DNA hydrogels were concentrated in the dextran-rich droplets, whereas short ssDNA were distributed in both dextran-rich droplets and a PEG-rich continuous phase. [20][21][22] We expected small ssDNA and large DNA structures would have different partitions and be separable by W/W-droplet fractionation (Figures 1B and 1 C). Therefore, the W/W-droplet system is expected to be applied to purify DNA structures such as DNA motifs, DNA origami, DNA hydrogel, and DNA microtubes.…”