基于柱温调节优化环状 RNA 的 SEC-HPLC 分析纯化工艺

Optimization of SEC-HPLC Analytical and Purification Workflow for Circular RNA via Column Temperature Regulation

关键词:  环状RNA  体积排阻高效液相色谱  柱温  纯化

Key words:  circular RNA  SEC-HPLC  column temperature  purification

摘要:

目的 调节柱温优化适用于环状 RNA（circular RNA, circRNA）的尺寸排阻高效液相色谱（SEC-HPLC）纯化工艺，探究柱温对两类编码型 circRNA 色谱行为、分离分辨率、产物纯度、收率及 RNA 完整性的影响，筛选最优温控纯化参数，并对比纯化前后 circRNA 体外翻译活性。方法 以circRNA-EGFP、circRNA-Fluc 两种环化粗样品为研究对象，采用 Sepax SEC-2000 Å 体积排阻色谱柱，设置5、25、37、55、70 ℃五档柱温开展 SEC-HPLC 分离；记录不同温度下色谱峰形与组分保留时间，计算线性前体与目标 circRNA 分离度 Rs，分析纯化收率；收集各温度 circRNA 洗脱组分，琼脂糖凝胶电泳分析 circRNA 纯度；将 SEC-HPLC 纯化产物与未纯化粗产物分别转染 293T 细胞，测定EGFP荧光强度、萤火虫荧光素酶表达水平评价生物活性。结果 随柱温升高，所有组分保留时间同步缩短。5 ℃低温下 RNA 二级结构稳定，线性杂质与 circRNA 色谱峰大面积重叠，分离度最低；25、37 ℃升温后，线性前体与 circRNA 可初步分离，分离度与产物纯度同步提升；55 ℃可进一步提升综合纯化效果，实现线性前体、nicked RNA与目标 circRNA的分离，circRNA电泳纯度达到各组最高；70 ℃强高温未能提升分离度，反而会生成降解产物，完整 circRNA 得率显著下降。两类编码 circRNA 随温度变化的色谱、纯度变化规律基本一致。细胞实验证实，SEC-HPLC纯化产物转染后的细胞内 EGFP 荧光强度、萤火虫荧光素酶活性均显著优于未纯化粗样品。结论 柱温通过调控 RNA 二级结构变性程度改变流体力学尺寸差异，影响 circRNA SEC-HPLC 分离纯化效果；本实验体系最优纯化柱温为 55 ℃，可同时获得高分离分辨率、高纯度和可接受收率的circRNA；该温控 SEC-HPLC 工艺操作简便、对不同编码序列 circRNA 兼容性良好，可为编码型 circRNA 的制备与质量控制提供参考。

Abstract:

OBJECTIVE To optimize the size-exclusion high-performance liquid chromatography (SEC-HPLC) purification process for circular RNA (circRNA) by regulating column temperature, and to investigate the effects of column temperature on chromatographic behavior, separation resolution, product purity, recovery yield and RNA integrity of two coding circRNAs. The optimal temperature-controlled purification parameters were screened, and the in vitro translational activity of circRNA before and after purification was compared. METHODS Two crude circularized samples, circRNA-EGFP and circRNA-Fluc, were used as research materials. Sepax SEC-2000 Å size-exclusion chromatographic column was adopted to perform SEC-HPLC separation at five gradient column temperatures of 5, 25, 37, 55 and 70 °C. The chromatographic peak profiles and component retention time at each temperature were recorded, the resolution (Rs) between linear precursors and target circRNA was calculated, and the main peak purity and purification recovery were statistically analyzed. Eluted circRNA fractions collected at each temperature were subjected to agarose gel electrophoresis to analyze circRNA purity and detect nicked degraded RNA fragments. SEC-HPLC-purified products and unpurified crude samples were separately transfected into 293T cells, and EGFP fluorescence intensity as well as firefly luciferase expression level were determined to evaluate biological activity. RESULTS The retention time of all components shortened synchronously as column temperature increased. At low temperature of 5 °C, RNA secondary structure remained stable, leading to extensive peak overlap between linear impurities and circRNA with the lowest resolution. After temperature elevation to 25 °C and 37 °C, linear precursors and circRNA could be preliminarily separated, accompanied by simultaneous improvement of resolution and product purity. Column temperature at 55 °C further improved the comprehensive purification performance, realizing effective separation of linear precursors, nicked RNA and target circRNA, and the electrophoretic purity of circRNA reached the maximum among all groups. High temperature of 70 °C failed to enhance separation resolution, instead triggered massive generation of degraded fragments and drastically reduced the yield of intact circRNA. The two coding circRNAs presented nearly consistent variation trends in chromatographic profile and product purity along with temperature changes. Cellular assays verified that intracellular EGFP fluorescence intensity and firefly luciferase activity of SEC-HPLC-purified circRNA were significantly higher than those of unpurified crude samples. CONCLUSION Column temperature alters the discrepancy of hydrodynamic volume by regulating the denaturation degree of RNA secondary structure, thereby determining the purification performance of circRNA in SEC-HPLC system. The optimal column temperature for purification in this experimental system is 55 °C, which can simultaneously obtain circRNA with high separation resolution, high purity and acceptable recovery yield. This temperature-controlled SEC-HPLC protocol features simple operation and favorable compatibility with circRNAs carrying different coding sequences, which can provide references for the preparation and quality control of coding circRNA.