Active Uptake and Trafficking of Nucleoside Triphosphates In Vivo

Abstract

Modified nucleoside triphosphates (NTPs) are powerful probes and medicines, but their anionic character impedes membrane permeability. As such, invasive delivery techniques, transport carriers, or prodrug strategies are required for their in vivo use. Here, we present a fluorescent 2′-deoxyribonucleoside triphosphate “TAMRA-dATP” that exhibits surprisingly high bioavailability in vivo. TAMRA-dATP spontaneously forms nanoparticles in Mg+2-containing buffers that are taken into the vesicles of living cells and animals by energy-dependent processes. In cell cultures, photochemical activation with yellow laser light (561 nm) facilitated endosomal escape of TAMRA-dATP, resulting in its metabolic incorporation into DNA in vitro. In contrast, in vivo studies revealed that TAMRA-dATP is extensively trafficked by active pathways into cellular DNA of zebrafish (Danio rerio) and Caenorhabditis elegans where DNA labeling was observed in live animals, even without photochemical release. Metabolic labeling of DNA in whole, living animals can therefore be achieved by simply soaking animals in a buffer containing TAMRA-dATP or a structurally related compound, Cy3-dATP.

Tropolone‐Conjugated DNA: A Fluorescent Thymidine Analogue Exhibits Solvatochromism, Enzymatic Incorporation into DNA and HeLa Cell Internalization

“…In the literature, native triphosphate nucleosides exhibit poor cell permeability [24] . Recently, Hocek et al.…”

“…In the literature, native triphosphate nucleosides exhibit poor cell permeability. [24] Recently, Hocek et al have reported the transfection of modified dNTP using a unique transporter, synthetic nucleoside triphosphate transporter (SNTT). [25] However, nucleosides and related molecules are prodrugs for various diseases including antiviral, anticancer, and antibiotic drugs.…”

In vivoquantitative high-throughput screening for drug discovery and comparative toxicology

“…In addition to phenotypic classifications possible from fluorescently tagged proteins or anatomical regions such as the gut, body wall, pharynx or neurons, the three-fluorescent channel capability of LSC would, in principle, enable colocalization and multiplexed assay formats ( Auld et al, 2006 ). For example, metabolic incorporation of TAMRA-dATP into genomic DNA or ‘cell painting’ could facilitate a range of phenotypic profiles in C. elegans ( Gustafsdottir et al, 2013 ; Schreier et al, 2022 ). Although we conducted the current study with LSC, using automated microscopy as a primary or LSC-coupled output is certainly feasible ( Gosai et al, 2010 ).…”

In vivo quantitative high-throughput screening for drug discovery and comparative toxicology

“…In addition to phenotypic classifications possible from fluorescently tagged proteins or anatomical regions such as the gut, body wall, phalanx, or neurons, the three fluorescent channel capability of LSC would in principle enable colocalization and multiplexed assay formats (Auld et al, 2006). For example, metabolic incorporation of TAMRA-dATP into genomic DNA or “cell painting” could facilitate a range of phenotypic profiles in C. elegans (Gustafsdottir et al, 2013; Schreier et al, 2022). While we conducted the current study with LSC, using automated microscopy as a primary or LSI-coupled output is certainly feasible (Gosai et al, 2010).…”