Fluorescent probe displacement assays reveal unique nucleic acid binding properties of human nudix enzymes

• Published in: Analytical biochemistry Vol. 595; p. 113622
• Main Authors: Ray, Atreyei, Frick, David N.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2020
• Subjects: 7-Methlyguanosine triphosphate hydrolase; Binding Sites; De-capping enzyme; Diphosphatase; DNA - chemistry; DNA Repair Enzymes - analysis; Escherichia coli - enzymology; Fluorescent Dyes - chemistry; Humans; NADH pyrophosphatase; Nudix; Nudix Hydrolases; Orf257; Phosphoric Monoester Hydrolases - analysis; Pyrophosphatases - analysis; Recombinant Proteins - analysis; RNA - chemistry; NADH pyrophosphatase; Orf257; Nudix; Diphosphatase; 7-Methlyguanosine triphosphate hydrolase; De-capping enzyme
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113622

Nudix proteins are members of a large family of homologous enzymes that hydrolyze nucleoside diphosphates linked to other compounds. The substrates for a subset of Nudix enzymes are all nucleotides linked to RNA, like the m7G mRNA caps and the more recently discovered NAD(H) RNA caps. However, the RNA affinity and nucleic acid specificity of Nudix proteins has not yet been explored in depth. In this study we designed new fluorescence-based assays to examine the interaction of purified recombinant E. coli NudC and human Nudt1 (aka MTH1) Nudt3, Nudt12, Nudt16, and Nudt20 (aka Dcp2). All Nudix proteins except Nudt1 and Nudt12 bound both RNA and DNA stoichiometrically with high affinity (dissociation constants in the nanomolar range) and no clear sequence specificity. In stark contrast, Nudt12 binds RNA but not similar DNA oligonucleotides. Nudt12 also bound RNAs with 5’ NAD+ caps more tightly than those with NADH or m7G cap. NudC was similarly selective against m7G caps but did not differentiate between NAD+ and NADH capped RNA. Nudt3, Nudt16, and Nudt20 bound m7G capped RNA more tightly than RNA with NADH caps. [Display omitted] •Simple, sensitive, fluorescence-based nucleic acid bindings assays for identifying Nudix protein ligands.•Several human Nudix proteins bind nucleic acids with dissociation constants in the low nanomolar range.•The human Nudt12 protein specifically binds RNA capped with nicotinamide adenine dinucleotide.•The human Nudt3, Nudt16, and Nudt20 (Dcp2) proteins bind both DNA and RNA but prefer to bind m7G-capped RNA.