RNAi Therapeutics, NIBR Biologics Center, Novartis Institutes for BioMedical Research (NIBR), Inc., 250 Massachusetts Avenue, Cambridge, MA 02139, USA.
Rapid Commun Mass Spectrom. 2011 Nov 15;25(21):3207-15. doi: 10.1002/rcm.5221.
Traditional methods for extracting oligonucleotides from serum and other biological fluids are often time-consuming and require multiple steps. Magnetic particle based separation of oligonucleotides has gained importance recently due to the advantages of simplicity and high efficiency. Here we report the development and optimization of commercially available strong anion-exchange (SAX) magnetic beads for the extraction of siRNA from human serum. The beads allowed for rapid extraction of siRNA from human serum in 100-200 μL of liquid chromatography/mass spectrometry (LC/MS)-compatible buffer in less than 1 h for a 96-well plate with no further drying steps. Due to the strong cation-binding properties of oligonucleotides, volatile ammonium salts such as triethylammonium bicarbonate (TEAB), ammonium bicarbonate, and NH(4) Cl were used to elute the siRNA from the beads. For more hydrophobic siRNA sequences, the addition of 5-10% organic solvent was required for elution. The recovery of chemically modified siRNA from human serum was around 80% for two types of beads examined; however, the recovery for highly modified sequences differed greatly between the two types of beads. In addition to extracting highly modified oligonucleotides, the SAX beads were also able to extract liposomal formulated siRNAs from serum with no interference from the lipid formulation. The extraction of siRNA from human serum was linear over the tested range of 50 ng/mL to 10 µg/mL. Using this extraction methodology, we have created a workflow to monitor siRNA serum stability by LC/MS. Initial observations confirm that RNase A type degradation with strand cleavage on the 3' side of uridine or cytosine is the dominant cleavage pattern in serum. This finding has implications for the selection and modification of therapeutic siRNAs and demonstrates the utility of magnetic beads as a simple and rapid extraction technique for siRNA.
传统的从血清和其他生物液体中提取寡核苷酸的方法通常耗时且需要多个步骤。基于磁性颗粒的寡核苷酸分离由于其简单和高效的优点，最近得到了重视。在这里，我们报告了商业上可获得的强阴离子交换（SAX）磁性珠的开发和优化，用于从人血清中提取 siRNA。这些珠子允许在不到 1 小时的时间内，从 96 孔板中以 100-200 μL 的液相色谱/质谱（LC/MS）兼容缓冲液中快速提取 siRNA，而无需进一步干燥步骤。由于寡核苷酸具有强烈的阳离子结合特性，因此使用挥发性铵盐，如三乙基碳酸氢铵（TEAB）、碳酸氢铵和 NH4Cl 从珠子上洗脱 siRNA。对于更疏水的 siRNA 序列，需要添加 5-10%的有机溶剂进行洗脱。从两种类型的珠子上提取化学修饰的 siRNA 的回收率约为 80%；然而，两种类型的珠子之间高度修饰的序列的回收率有很大差异。除了提取高度修饰的寡核苷酸外，SAX 珠还能够从血清中提取脂质体配方的 siRNA，而不会受到脂质配方的干扰。从人血清中提取 siRNA 在 50ng/mL 至 10μg/mL 的测试范围内呈线性。使用这种提取方法，我们创建了一种通过 LC/MS 监测 siRNA 血清稳定性的工作流程。初步观察证实，在血清中，RNA 酶 A 型降解并在尿嘧啶或胞嘧啶的 3'侧产生链断裂是主要的断裂模式。这一发现对治疗性 siRNA 的选择和修饰具有重要意义，并证明了磁性珠作为一种简单快速的 siRNA 提取技术的实用性。
