Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization
Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization
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DOI：
10.1039/c7sm02146a
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发表时间：
2018-01-21
期刊：
影响因子：
3.4
通讯作者：
Keating, C. D.
中科院分区：
文献类型：
--
作者：
Marianelli, A. M.;Miller, B. M.;Keating, C. D.
本文报道了中性大分子聚乙二醇（PEG）（8 kDa）和Ficoll（70 kDa）对聚尿苷酸（polyU）/精胺复合凝聚体系液-液相分离的影响。PEG的加入降低了相分离所需的精胺量和凝聚温度（T-c）。我们解释这些相行为的影响所产生的排斥体积和优先的相互作用的二级结构/精胺相关的polyU分子的凝聚和可溶性polyU/精胺polyU复合物的关联，形成凝聚液滴。在存在荧光标记的PEG或Ficoll拥挤物的情况下形成的凝聚物的检查表明，Ficoll积聚，而PEG被排除在凝聚物相中，这提供了对相行为差异的进一步了解。拥挤剂影响生物分子溶质的分布：荧光标记的U15 RNA寡聚体分配到polyU/精胺凝聚体中的量通过20重量%的Ficoll 70 kDa增加约两倍，并且通过20重量%的PEG 8 kDa增加超过两个数量级。凝聚相的体积在拥挤的存在下相对于稀释的缓冲溶液减少。这些研究结果表明，大分子拥挤的相行为和溶质分配的潜在影响，应考虑在模型系统的细胞内无膜细胞器。
We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid-liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (T-c). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.