Homchan Aimorn, Patchsung Maturada, Chantanakool Pheerawat, Wongsatit Thanakrit, Onchan Warunya, Muengsaen Duangkamon, Thaweeskulchai Thana, Tandean Martin, Sakpetch Theeradon, Suraritdechachai Surased, Aphicho Kanokpol, Panchai Chuthamat, Taiwan Siraphob, Horthongkham Navin, Sudyoadsuk Taweesak, Reinhardt Aleks, Uttamapinant Chayasith
School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong 21210, Thailand.
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
J Am Chem Soc. 2025 Mar 26;147(12):10088-10103. doi: 10.1021/jacs.4c11893. Epub 2025 Feb 13.
Isothermal techniques for amplifying nucleic acids have found extensive applications in genotyping and diagnostic tests. These methods can be integrated with sequence-specific detection strategies, such as CRISPR-based detection, for optimal diagnostic accuracy. In particular, recombinase-based amplification uses proteins from the Escherichia virus T4 recombination system and operates effectively at moderate temperatures in field and point-of-care settings. Here, we discover that recombinase polymerase amplification (RPA) is controlled by liquid-liquid phase separation, where the condensate formation enhances the nucleic acid amplification process. While two protein components of RPA could act as scaffold proteins for condensate formation, we identify T4 UvsX recombinase as the key regulator orchestrating distinct core-shell arrangements of proteins within multiphase condensates, with the intrinsically disordered C-terminus of UvsX being crucial for phase separation. We develop volumetric imaging assays to visualize RPA condensates and the reaction progression in whole volumes, and begin to dissect how macroscopic properties such as size distribution and droplet count could contribute to the overall reaction efficiency. Spatial organization of proteins in condensates may create optimal conditions for amplification, and disruption of such structures may diminish the amplification efficiency, as we demonstrate for the case of reverse transcription-RPA. The insight that RPA functions as a multiphase condensate leads us to identify the UvsX mutant, which has a distinct phase-separation propensity compared to the wild-type enzyme and can enhance RNA detection via RPA-coupled CRISPR-based diagnostics.
用于扩增核酸的等温技术已在基因分型和诊断测试中得到广泛应用。这些方法可与序列特异性检测策略相结合，如基于CRISPR的检测，以实现最佳诊断准确性。特别是，基于重组酶的扩增利用来自大肠杆菌病毒T4重组系统的蛋白质，在现场和即时护理环境的适度温度下有效运行。在这里，我们发现重组酶聚合酶扩增（RPA）受液-液相分离控制，其中凝聚物的形成增强了核酸扩增过程。虽然RPA的两种蛋白质成分可作为凝聚物形成的支架蛋白，但我们确定T4 UvsX重组酶是协调多相凝聚物中蛋白质不同核壳排列的关键调节因子，UvsX内在无序的C末端对相分离至关重要。我们开发了体积成像分析方法，以可视化RPA凝聚物和整个体积中的反应进程，并开始剖析诸如大小分布和液滴数量等宏观特性如何影响整体反应效率。凝聚物中蛋白质的空间组织可能为扩增创造最佳条件，破坏这种结构可能会降低扩增效率，正如我们在逆转录-RPA的案例中所证明的那样。RPA作为多相凝聚物发挥作用这一见解使我们鉴定出UvsX突变体，与野生型酶相比，它具有独特的相分离倾向，并且可以通过基于RPA耦合CRISPR的诊断增强RNA检测。
