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 H 2 O 2 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.

DNA Modifications Enabling Proximity Biotinylation

“…We have previously shown that peroxidase-based biotinylation of oligodeoxyribonucleotides with BT is enabled by DNA conjugation with 5’ fluorescein and we described structural details of BT-fluorescein adducts [6]. We demonstrated that an in vitro streptavidin gel shift assay is capable of detecting biotinylation of both RNA and 5’-fluorescein-modified DNA oligonucleotides and, importantly, provided molecular insights into differences in RNA and DNA biotinylation.…”

“…To determine if other biological functional groups are reactive with BT phenoxy radicals, we devised a competition assay for the assessment of all other natural amino acids. In this assay, a streptavidin gel shift is used to detect horseradish peroxidase (HRP)and H 2 O 2 -dependent radical biotinylation of 5’-fluorescein modified oligodeoxyribonucleotide LJM-6132 [6]. Increasing concentrations of individual competing amino acids are added to the reaction.…”

“…APEX and APEX2, on the other hand, catalyze production of short-lived reactive phenoxy radical intermediates from biotin tyramide (BT) in the presence of H 2 O 2 . Though proximity biotinylation by peroxidases has the impressive advantage of dual protein and RNA radical biotinylation, this approach cannot be used directly for biotinylation of unmodified DNA [6].…”

DNA Modifications Enabling Proximity Biotinylation

“…BioID and TurboID Biotin ligases catalyze synthesis of a highly reactive biotinyl-5′-adenylate intermediate from biotin and ATP, allowing proximity biotinylation of primary amine nucleophiles as reactive targets. ,, While such targets exist in proteins on lysine residues and N-termini, none are found in nucleic acids. APEX and APEX2, on the other hand, catalyze production of short-lived reactive phenoxy radical intermediates from biotin tyramide (BT) in the presence of H 2 O 2 . , Though proximity biotinylation by peroxidases has the impressive advantage of dual protein and RNA radical biotinylation, this approach cannot be used directly for biotinylation of unmodified DNA …”

“…It is known that tyrosine residues are protein reactive sites biotinylated by APEX2-generated BT phenoxy radicals. , To determine if other biological functional groups are reactive with BT phenoxy radicals, we devised a competition assay for the assessment of all other natural amino acids. In this assay, a streptavidin gel shift is used to detect horseradish peroxidase (HRP) and H 2 O 2 -dependent radical biotinylation of 5′-fluorescein-modified oligodeoxyribonucleotide 6132 . Increasing concentrations of individual competing amino acids are added to the reaction.…”