BellBrook Labs, Madison, WI, USA.
Thermo Fisher Scientific, Madison WI, USA.
SLAS Discov. 2024 Jun;29(4):100161. doi: 10.1016/j.slasd.2024.100161. Epub 2024 May 22.
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 K 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.
蛋白质和核酸的甲基化在表观遗传调控中起着基本作用，而寻找甲基转移酶 (MT) 抑制剂是一个非常活跃的研究领域。由于 MT 及其产物的多样性，检测 S-腺苷同型半胱氨酸 (SAH) - S-腺苷甲硫氨酸 (SAM) 依赖性甲基化反应的不变产物的测定方法比检测特定甲基化事件的方法具有一些优势。然而，直接、均相检测 SAH 需要一种能够区分 SAH 和 SAM 的试剂，SAM 仅在一个甲基组上有所不同。此外，MT 是缓慢的酶，许多 MT 对 SAM 的亲和力为亚微摩尔级；这些特性转化为在存在过量 SAM 的情况下需要在低纳摩尔浓度下检测 SAH。为了满足这些需求，我们利用了天然存在的 SAH 感应 RNA 适体（或核酶）的精湛分子识别特性。通过将核酶分割成两个片段，使得 SAH 结合诱导三聚体复合物的组装，我们设计了传感器，将 SAH 的结合转化为正荧光偏振（FP）和时间分辨荧光共振能量转移（TR-FRET）信号。这种分裂的核酶结构，称为 AptaFluor™ SAH 甲基转移酶测定法，允许在浓度低于 10 nM 的情况下对 SAH 进行强大的检测（Z' > 0.7），并且在存在典型的 MT 测定成分的情况下，信号稳定性可维持过夜。AptaFluor 测定法可耐受多种 MT 底物，包括组蛋白、核小体、DNA 和 RNA，并且我们证明了它作为具有 SAM K 值 <1µM 的几种甲基转移酶的强大酶测定法的实用性。通过对 1,280 种化合物对 SARS-CoV-2 RNA 加帽酶 nsp14 进行先导筛选，对 AptaFluor 测定法进行了高通量筛选 (HTS) 的验证。通过在低纳摩尔浓度下直接、均相检测 SAH，AptaFluor 测定法为在生理相关的 SAM 浓度下筛选和分析 MT 提供了通用平台。
