PDF(Pubmed)
Abstract：
Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z\' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.
摘要：
蛋白质和核酸的甲基化在表观遗传调控中起着重要作用。甲基转移酶（MT）抑制剂的发现是一个活跃的领域。由于MTs及其产品的多样性，与检测特定甲基化事件的方法相比,检测S-腺苷甲硫氨酸(SAM)依赖性甲基化反应的不变产物S-腺苷同型半胱氨酸(SAH)的测定方法具有一些优势.然而,直接，SAH的均相检测需要能够区分SAH和SAM的试剂，不同的是一个甲基。此外，MT是缓慢的酶，许多对SAM具有亚微摩尔亲和力；这些特性转化为需要在过量SAH的存在下以低纳摩尔浓度检测SAH。为了满足这些需求,我们利用了天然存在的SAH敏感RNA适体的精致分子识别特性，或者核糖开关。把核糖开关分成两个片段,这样SAH结合诱导三聚体复合物的组装，我们设计了传感器，将SAH的结合转换为正荧光偏振（FP）和时间分辨的Förster共振能量转移（TR-FRET）信号。分裂核糖开关配置，称为AptaFluor™SAH甲基转移酶测定，允许在低于10nM的浓度下可靠地检测SAH(Z'>0.7)，在典型的MT测定组分存在下具有过夜信号稳定性。AptaFluor测定法耐受不同的MT底物，包括组蛋白,核小体,DNA和RNA,我们证明了它的实用性，SAMKm值<1µM的几种甲基转移酶的酶法检测。通过对针对SARS-CoV-2RNA加帽酶的1,280种化合物进行先导筛选，对HTS进行了验证。nsp14.通过启用直接，在低纳摩尔浓度下均匀检测SAH，AptaFluor分析为在生理相关SAM浓度下筛选和分析MTs提供了通用平台.
