PO.ET02.09 · 实验与分子治疗
一种利用未修饰RNA检测XRN1核酸外切酶活性的HTS检测法
An HTS assay for detection of XRN1 exoribonuclease activity with unmodified RNA
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
XRN1是一种5'到3'的核酸外切酶，可将单链和双链RNA降解为核苷酸5'单磷酸产物。它在mRNA周转和先天免疫中发挥重要作用，通过抑制胞质RNA传感器（例如PKR和MDA5）的激活来实现。XRN1的缺失在癌细胞培养模型中与高干扰素信号相关，使其成为癌症免疫治疗的有吸引力的靶点。在此，我们描述了使用Transcreener AMP 2/GMP 2检测法开发用于筛选和分析抑制剂的HTS兼容检测方法。现有的RNA酶检测法依赖荧光标记的RNA，这对可使用的底物施加了限制，并可能影响酶的结合或催化。使用Transcreener AMP 2/GMP 2检测法，XRN1产生的嘌呤单磷酸通过竞争性免疫检测直接检测，采用远红色荧光偏振（FP）或时间分辨荧光共振能量转移（TR-FRET）读数。这种形式是均相的（混合即读），与多种RNA底物兼容，并且抗筛选化合物的干扰。使用在BaV感染的昆虫细胞中生产的全长人XRN1，我们测试了多种RNA底物，包括具有不同末端（平末端、突出末端等）的合成双链和单链寡核苷酸以及总酵母RNA。观察到的AMP/GMP形成速率范围为0.054 sec⁻¹至0.52 sec⁻¹，单链RNA的活性高于双链底物；polyA用于更详细的研究。虽然poly-A的Km与长度无关，但100聚体的降解速率和程度高于300聚体。在剂量反应实验中测试了一种探针抑制剂pAp，得出IC50为114.8 nM，这与使用荧光标记的DNA/RNA杂交体测量的值吻合良好。通过筛选1280种生物活性物质的集合对该检测进行了HTS验证，得出Z'>0.9，并鉴定出在剂量反应实验中得到确认的命中物。
查看英文原文 English abstract
XRN1 is a 5' to 3' exoribonuclease that degrades both single- and double-stranded RNA to nucleotide 5' monophosphate products. It plays an important role in mRNA turnover and in innate immunity, by damping activation of cytosolic RNA sensors, e.g., PKR and MDA5. Loss of XRN1 has been linked to high interferon signaling in cancer cell culture models, making it an attractive target for cancer immunotherapy. Here we describe development of HTS-compatible assays for screening and profiling inhibitors using the Transcreener AMP 2 /GMP 2 Assay. Existing assays for RNAses rely on fluorescently labeled RNAs, which impose limitations on the substrates that can be used and may affect enzyme binding or catalysis. WIth the Transcreener AMP 2 /GMP 2 Assay, purine monophosphates produced by XRN1 are directly detected by a competitive immunoassay with a far-red, fluorescence polarization (FP) or time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. This format is homogenous (mix-and-read), compatible with diverse RNA substrates, and resistant to interference from screening compounds. Using full length human XRN1 produced in BaV-infected insect cells, we tested a number of RNA substrates, including synthetic double and single strand oligos with different ends (blunt, overhang, etc.) as well as total yeast RNA. Rates of AMP/GMP formation ranging from 0.054 sec -1 to 0.52 sec -1 were observed, with single strand RNAs yielding higher activity than double strand substrates; polyA was used for more detailed studies. Though the K m for poly-A was not length-dependent, the rate and extent of degradation was higher for a 100-mer than a 300-mer. A probe inhibitor, pAp, was tested in dose response experiments yielding an IC 50 of 114.8 nM, which aligns well with the value measured using a fluorescently labeled DNA/RNA hybrid. The assays were validated for HTS by screening a collection of 1280 bio-actives, yielding a Z'> 0.9 and identification of hits that were confirmed in dose response experiments.
利益披露 Disclosure
M. Ali, None..
R. Lowery, None.