Quantification of all 12 canonical ribonucleotides by real-time fluorogenic in vitro transcription.

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Ribonucleotides*/analysis ; Biochemistry*/methods; Diphosphates ; Nucleotides/chemistry

Enzymatic methods to quantify deoxyribonucleoside triphosphates have existed for decades. In contrast, no general enzymatic method to quantify ribonucleoside triphosphates (rNTPs), which drive almost all cellular processes and serve as precursors of RNA, exists to date. ATP can be measured with an enzymatic luminometric method employing firefly luciferase, but the quantification of other ribonucleoside mono-, di-, and triphosphates is still a challenge for a non-specialized laboratory and practically impossible without chromatography equipment. To allow feasible quantification of ribonucleoside phosphates in any laboratory with typical molecular biology and biochemistry tools, we developed a robust microplate assay based on real-time detection of the Broccoli RNA aptamer during in vitro transcription. The assay employs the bacteriophage T7 and SP6 RNA polymerases, two oligonucleotide templates encoding the 49-nucleotide Broccoli aptamer, and a high-affinity fluorogenic aptamer-binding dye to quantify each of the four canonical rNTPs. The inclusion of nucleoside mono- and diphosphate kinases in the assay reactions enabled the quantification of the mono- and diphosphate counterparts. The assay is inherently specific and tolerates concentrated tissue and cell extracts. In summary, we describe the first chromatography-free method to quantify ATP, ADP, AMP, GTP, GDP, GMP, UTP, UDP, UMP, CTP, CDP and CMP in biological samples.
(© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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Samfundet Folkhälsan; Jane and Aatos Erkko Foundation; Medicinska Understödsföreningen Liv och Hälsa; University of Helsinki

0 (Diphosphates)
0 (Nucleotides)
0 (Ribonucleotides)

Date Created: 20231126 Date Completed: 20240125 Latest Revision: 20240125

20240125

PMC10783517

10.1093/nar/gkad1091

38008466