Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Satusky, Matthew; Johnson, Caitlin V.; Erie, Dorothy A.

doi:10.1016/j.bpj.2023.02.011

Biophysical Journal

2023

DOI: 10.1016/j.bpj.2023.02.011

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Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Matthew Satusky

Caitlin V. Johnson

Dorothy A. Erie

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2024

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Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method

He,

Xiang

2024

Anal. Chem.

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In nature, DNA phosphorothioate (PT) is found in the genomic materials of some prokaryotes. In contrast, whether there is natural RNA PT is still a question under debate. A groundbreaking study reported the discovery of RNA PT in cellular RNA samples from both prokaryotes and eukaryotes at contents of >100 PT per million nucleotides (PPM-nt) according to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. However, this finding was challenged by a later work showing that other RNA modifications, such as 2′-O-methylation, could give almost the same LC-MS/MS signal patterns as RNA PT. As the LC-MS/MS technique led to contradicting conclusions, another independent method is thus needed to interrogate the presence of RNA PT in nature. In this work, we have developed a highly selective and sensitive fluorescence method for RNA PT quantification based on a new RNA PT-specific conversion reaction. It can detect as low as 2.8 PPM-nt in RNA without interference from RNA thiobases or protein cysteines. We measured the total RNA samples from some bacteria and human cells using this method. None of these samples gave any RNA PT signal above the detection limit (2.8 PPM-nt), suggesting that the widespread presence of natural RNA PT at the 100 PPM-nt level or above is highly unlikely. Nevertheless, due to the limited number of cell species tested in this work, the possible existence of natural RNA PT cannot be excluded. The fluorescence method reported here is simple and low-cost; therefore, it should be an ideal assay for broadly screening various types of cells to search for the clue of RNA PT in nature.

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Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method

He,

Xiang

2024

Anal. Chem.

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Rapid, inexpensive, sequence-independent fluorescent labeling of phosphorothioate DNA

Cited by 1 publication

References 38 publications

Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method

Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method

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