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Regulation of Monovalent/Divalent Cation Concentration on Liquid-Liquid Phase Separation of Protein-Nucleic Acid
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摘要: 蛋白质和核酸的液液相分离（liquid-liquid phase separation, LLPS）对无膜细胞器的形成起着重要作用. 在病理研究中，LLPS也可以导致蛋白质的异常聚集从而引起某些神经退行性疾病的发生. 因此研究蛋白质-核酸LLPS的有效调控，能够对理解生物结构功能以及相关疾病治疗提供一定的参考价值. 主要利用光学显微镜、动态光散射仪以及紫外分光光度计，研究一价/二价阳离子对多聚赖氨酸（poly-L-lysine, PLL）-脱氧核糖核酸（DNA）LLPS的调控，并对其机制进行分析. 试验中观察到PLL-DNA的LLPS会随着钠/钾离子浓度的升高而逐渐形成沉淀，从出现沉淀到沉淀最多时，钠/钾离子浓度大约为100 mmol/L到 600 mmol/L. 在钠离子浓度高于600 mmol/L时聚合物沉淀开始溶解，再次有LLPS发生. 然而镁/钙离子对于PLL-DNA的聚合物的调控表现出更高的效率，形成沉淀的镁/钙离子临界浓度为50 mmol/L，重新发生LLPS的浓度大约为300 mmol/L. 通过对该溶液的吸光度进行分析，得到的结论与显微镜观察一致，并且该溶液的电泳迁移率随着一价/二价阳离子浓度的升高出现了逆转现象，即电泳值由负变正，说明一价/二价阳离子对蛋白质-核酸LLPS的调控与电荷变化有关，二价阳离子对其调控效率高于一价阳离子. 最后，绘制了一种聚合物的结构模型，并为这一相分离过程提供合理的解释.Abstract: The liquid-liquid phase separation (LLPS) of proteins and nucleic acids plays an important role in the formation of membrane-free organelles. In pathological studies, the LLPS can also lead to the abnormal accumulation of proteins and lead to some neurodegenerative diseases. Therefore, the study of the effective regulation of the LLPS of protein-nucleic acid can provide some reference value for us to understand the biological structure and function and the treatment of related diseases. Optical microscope, dynamic light scattering and ultraviolet spectrophotometers were used to study the regulation of the LLPS of poly-L-lysine (PLL)-deoxyribonucleic acid (DNA) by monovalent/divalent cations. The formation mechanism of LLPS phenomenon of PLL-DNA was also analyzed. It was observed that the concentration of sodium/potassium ion was higher than 100 mmol/L, the LLPS of PLL-DNA precipitated gradually and precipitated mostly at 600 mmol/L. When the concentration of sodium/potassium ion was higher than 600 mmol/L, the polymer precipitation began to dissolve, and LLPS occured again. However, magnesium/calcium ion showed a higher efficiency in the regulation of PLL-DNA polymers. The critical concentration of magnesium/calcium ion to form precipitation was 50 mmol/L, and the concentration of re-occurrence of LLPS was about 300 mmol/L. The absorbance of the solution was analyzed and the same conclusions were obtained as observed in the microscopic experiment. The electrophoretic mobility of the solution was reversed that the value change from negative to positive with the increase of the concentration of monovalent/divalent cations, indicating that the regulation of monovalent and divalent cations on protein-nucleic acid LLPS is related to the change of charge, and the regulation by divalent cations is also more efficient than monovalent cations. Finally, a structural model of the polymer was proposed which provides a reasonable explanation for the phase separation process.
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Key words:
- DNA /
- poly-L-lysine /
- monovalent cation /
- divalent cation /
- liquid-liquid phase separation /
- electrostatic shielding
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图 1 氯化钠、PLL与DNA溶液的相互作用
(a) (b) 50 mmol/L NaCl，2 μg/μL PLL，(c) (d) 50 mmol/L NaCl，2 μg/μL DNA，(e) (f) 2 μg/μL PLL，2 μg/μL DNA
Figure 1. Interaction of sodium chloride, PLL and DNA solution
(a) (b) 50 mmol/L NaCl, 2 μg/μL PLL, (c) (d) 50 mmol/L NaCl, 2 μg/μL DNA, (e) (f) 2 μg/μL PLL, 2 μg/μL DNA
图 2 一价/二价阳离子对PLL-DNA聚合物的影响，其中PLL和DNA的质量浓度始终为2 μg/μL
（a）~（e）在PLL-DNA溶液中加入NaCl溶液后的显微镜成像（其中NaCl浓度分别为10、100、300、600、800 mmol/L），（f）~（j）在PLL-DNA溶液中加入KCl溶液后的显微镜成像（其中KCl浓度分别为10、100、300、600、800 mmol/L），（k）~（o）在PLL-DNA溶液中加入MgCl2溶液后的显微镜成像（其中MgCl2浓度分别为2、50、150、300、500 mmol/L），（p）~（t）在PLL-DNA溶液中加入CaCl2溶液后的显微镜成像（其中CaCl2浓度分别为2、50、150、300 、500 mmol/L），溶液温度保持在35 ℃左右
Figure 2. Experimental observation of monovalent/divalent cations on PLL-DNA polymers, concentrations of PLL and DNA: 2 μg/μL
(a)~(e) microscopic images after addition of NaCl solution to PLL-DNA solution (concentration of NaCl of 10, 100, 300, 600 and 800 mmol/L respectively), (f)~(j) microscopic images after addition of KCl to PLL-DNA solution (concentration of KCl of 10, 100, 300, 600 and 800 mmol/L respectively), (k)~(o) microscopic images after addition of MgCl2 to PLL-DNA solution (concentration of MgCl2 of 2, 50, 150, 300 and 500 mmol/L respectively), (p)~(t) microscopic images after addition of CaCl2 to PLL-DNA solution (concentration of CaCl2 of 2, 50, 150, 300 and 500 mmol/L respectively), temperature of solution: about 35 ℃
图 3 不同阳离子溶液中PLL-DNA的凝聚物的吸光度曲线图，其中PLL和DNA的浓度始终为2 μg/μL
（a）NaCl，（b）KCl，（c）MgCl2，（d）CaCl2
Figure 3. Absorbance change of PLL-DNA condensates in different cations solutions, concentration of PLL and DNA : 2 μg/μL
(a) NaCl, (b) KCl, (c)MgCl2, (d) CaCl2
图 4 外加电场下的PLL-DNA聚合物的电泳迁移率，其中PLL和DNA的浓度始终为1 ng/μL
（a）NaCl，（b）KCl，（c）MgCl2，（d）CaCl2
Figure 4. Electrophoretic mobility of PLL-DNA polymer under applied electric field, concentration of PLL and DNA: 1 ng/μL
(a) NaCl, (b) KCl, (c)MgCl2, (d) CaCl2
图 5 PLL-DNA凝聚物在一价/二价盐离子中的微观结构
（a）PLL-DNA溶液中的液滴结构，（b）加入一价阳离子后的PLL-DNA溶液中的沉淀结构，（c）加入二价阳离子后PLL-DNA溶液中的沉淀结构
Figure 5. Microstructure of PLL-DNA condensates in monovalent /divalent salt ions
(a) droplet structure in PLL-DNA solution, (b) precipitation structure of PLL-DNA solution after addition of univalent cations, (c) precipitation structure of PLL-DNA solution after adding divalent cations
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