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Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale.

ABSTRACT: Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA compounds. Nuclease degradation of the strands outside and inside the cell results in a misleading intracellular fluorescent signal. The signal is saturated by the fluorescence of the degradation product (phosphorylated dye). To test our hypothesis, we synthesized a range of DNA structures, including Cy3- and Cy5-labeled DNA cubes and DNA tetrahedra, and oligonucleotides with different stabilities toward nucleases. All give fluorescence signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control. Kinetics experiments revealed that uptake of stable DNA structures is delayed. We also studied several parameters influencing fluorescent data: stability of the DNA strand, fixation methods that can wash away the signal, position of the dye on the DNA strand, and design of FRET experiments. DNA nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility, programmability, and easy synthesis. However, few examples of successful DNA machines in vivo have been reported. We believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent dyes, in order to further propel the biological development, and application of DNA nanostructures.

SUBMITTER: Lacroix A

PROVIDER: S-EPMC6535766 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale.

Lacroix Aurélie A Vengut-Climent Empar E de Rochambeau Donatien D Sleiman Hanadi F HF

ACS central science 20190426 5

Fluorescent dye labeling of DNA oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells. Here, we report that intracellular fluorescence, and even FRET signals, cannot be correlated with the cellular uptake of intact DNA structures. Live cell imaging revealed high colocalization of cyanine-labeled DNA oligos and nanostructures with phosphorylated small-molecule cyanine dyes, one of the degradation products from these DNA c ...[more]

PMID: 31139724

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Project description:Indocyanine green (IC-Green), the only FDA approved near-infrared (NIR) fluorophore for clinical use, is attractive to researchers for the development of targeted optical imaging agents by modification of its structure and conjugation to monoclonal antibodies (mAbs) or their fragments. IC-Green derivative, ICG-sulfo-OSu (ICG-sOSu), is frequently used for antibody conjugation. However, ICG-sOSu is amphiphilic and readily facilitates aggregation of mAbs that is not easily separable from the desired immunoconjugates. Complications originating from this behavior are frequently overlooked by researchers. This study examined detailed chemical and biological characteristics of an ICG-sOSu-labeled mAb, panitumumab, and provided a clinically applicable strategy to deliver a pure conjugation product. Size-exclusion high-performance liquid chromatography (SE-HPLC) analysis of conjugation reactions, performed at molar reaction ratios of ICG-sOSu: mAb of 5, 10, or 20, resulted in isolable desired ICG-sOSu-panitumumab conjugation product in 72%, 53%, and 19% yields, respectively, with the remainder consisting of high molecular weight aggregates (>150 kDa) 14%, 30%, and 51%, respectively. The HPLC-purified ICG-sOSu-panitumumab products were analyzed by native and SDS polyacrylamide gel electrophoresis (PAGE) followed by optical imaging. Results indicated that the interaction between ICG-sOSu and panitumumab was due to both covalent and noncovalent binding of the ICG-sOSu to the protein. Noncovalently bound dye in the ICG-sOSu-panitumumab conjugate products was removed by extraction with ethyl acetate to further purify the HPLC-isolated conjugation products. With conserved immunoreactivity, excellent target-specific uptake of the doubly purified bioconjugates was observed with minimal liver retention in athymic nude mice bearing HER1-expressing tumor xenografts. In summary, the preparation of well-defined bioconjugate products labeled with commercial ICG-sOSu dye is not a simple process and control of the conjugation reaction ratio and conditions is crucial. Furthermore, absolute purification and characterization of the products is necessitated prior to in vivo optical imaging. Use of validated and characterized dye conjugate products should facilitate the development of clinically viable and reproducible IC-Green derivative and other NIR dye mAb conjugates for optical imaging applications.

Dataset Information

Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale.

Publications

Uptake and Fate of Fluorescently Labeled DNA Nanostructures in Cellular Environments: A Cautionary Tale.

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