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Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

ABSTRACT: Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H₂ O₂ is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.

SUBMITTER: Wilbanks B

PROVIDER: S-EPMC8052304 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Wilbanks Brandon B Garcia Brian B Byrne Shane S Dedon Peter P Maher L James LJ

Chembiochem : a European journal of chemical biology 20210127 8

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H<sub>2</sub> O<sub>2</sub> is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phen ...[more]

PMID: 33368926

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Project description:Practical laboratory classes teaching molecular pharmacology approaches employed in the development of therapeutic strategies are of great interest for students of courses in Biotechnology, Applied Biology, Pharmaceutic and Technology Chemistry, Translational Oncology. Unfortunately, in most cases the technology to be transferred to learning students is complex and requires multi-step approaches. In this respect, simple and straightforward experimental protocols might be of great interest. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. microRNA therapeutics, and (b) on the employment of biomolecules of great interest in applied biology and pharmacology, i.e. peptide nucleic acids (PNAs). The aims of the practical laboratory were to determine: (a) the possible PNA-mediated arrest in RT-qPCR, to be eventually used to demonstrate PNA targeting of selected miRNAs; (b) the possible lack of activity on mutated PNA sequences; (c) the effects (if any) on the amplification of other unrelated miRNA sequences. The results which can be obtained support the following conclusions: PNA-mediated arrest in RT-qPCR can be analyzed in a easy way; mutated PNA sequences are completely inactive; the effects of the employed PNAs are specific and no inhibitory effect occurs on other unrelated miRNA sequences. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). On the other hand, these laboratory lessons require some facilities, the most critical being the availability of instruments for PCR. While this might be a problem in the case these instruments are not available, we would like to underline that determination of the presence or of a lack of amplified product can be also obtained using standard analytical approaches based on agarose gel electrophoresis.

Dataset Information

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Publications

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Similar Datasets

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