Fricke Riley, Knudson Isaac, Swenson Cameron Verdayne, Smaga Sarah, Schepartz Alanna
Department of Chemistry, University of California, Berkeley, CA, USA.
NSF Center for Genetically Encoded Materials (C-GEM), Berkeley, CA, USA.
Nat Protoc. 2025 May;20(5):1246-1274. doi: 10.1038/s41596-024-01086-9. Epub 2025 Jan 6.
Aminoacyl-tRNA synthetases (aaRSs) provide an essential functional link between an mRNA sequence and the protein it encodes. aaRS enzymes catalyze a two-step chemical reaction that acylates specific tRNAs with a cognate α-amino acid. In addition to their role in translation, acylated tRNAs contribute to non-ribosomal natural product biosynthesis and are implicated in multiple human diseases. In synthetic biology, the acylation of tRNAs with a non-canonical α-amino acid or, more recently, a non-α-amino acid monomer is a critical first step in the incorporation of these monomers into proteins, where they can be used for fundamental and applied science. These endeavors all demand an understanding of aaRS activity and specificity. Here, we describe a liquid chromatography-mass spectrometry assay that directly monitors aaRS activity by detecting the intact acyl-tRNA product. After a simple tRNA acylation reaction workup, acyl- and non-acyl-tRNA molecules are resolved by using ion-pairing reverse-phase chromatography, and their exact masses are determined by using high-resolution time-of-flight mass spectrometry. Our assay is fast and simple, quantifies reaction yields as low as 0.23% and can also be used on tRNAs acylated with flexizyme to detect products that are undetectable by using standard techniques. The protocol requires basic expertise in molecular biology, liquid chromatography-mass spectrometry and RNase-free techniques. This protocol takes ≥5 h to complete, depending on the number of samples.
氨酰-tRNA合成酶（aaRSs）在mRNA序列与其编码的蛋白质之间提供了至关重要的功能联系。aaRS酶催化一个两步化学反应，用同源α-氨基酸使特定的tRNA发生酰化。除了在翻译过程中的作用外，酰化的tRNA还参与非核糖体天然产物的生物合成，并与多种人类疾病有关。在合成生物学中，用非经典α-氨基酸或最近用非α-氨基酸单体对tRNA进行酰化，是将这些单体掺入蛋白质的关键第一步，在蛋白质中它们可用于基础科学和应用科学。所有这些研究都需要了解aaRS的活性和特异性。在此，我们描述了一种液相色谱-质谱分析方法，该方法通过检测完整的酰化tRNA产物直接监测aaRS活性。经过简单的tRNA酰化反应处理后，利用离子对反相色谱法分离酰化和非酰化的tRNA分子，并通过高分辨率飞行时间质谱法测定它们的精确质量。我们的分析方法快速简便，可定量低至0.23%的反应产率，还可用于检测用柔性酶酰化的tRNA，以检测用标准技术无法检测到的产物。该方案需要分子生物学、液相色谱-质谱和无核糖核酸酶技术方面的基本专业知识。根据样品数量，该方案需要≥5小时才能完成。
