The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides

Bartnicki, Filip; Bonarek, Piotr; Kowalska, E.; Strzałka, Wojciech

doi:10.1038/s41598-017-02432-6

Cited by 8 publications

(6 citation statements)

References 23 publications

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“…Selection of DNA aptamers was performed using the SELEX method as described previously ( 28 – 30 ) with minor modifications. Trimeric StrepTag-PCNA was used as the selection target.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex

Kowalska

Bartnicki

Fujisawa

et al. 2017

Nucleic Acids Research

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Proliferating cell nuclear antigen (PCNA) is a multifunctional protein present in the nuclei of eukaryotic cells that plays an important role as a component of the DNA replication machinery, as well as DNA repair systems. PCNA was recently proposed as a potential non-oncogenic target for anti-cancer therapy. In this study, using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method, we developed a short DNA aptamer that binds human PCNA. In the presence of PCNA, the anti-PCNA aptamer inhibited the activity of human DNA polymerase δ and ϵ at nM concentrations. Moreover, PCNA protected the anti-PCNA aptamer against the exonucleolytic activity of these DNA polymerases. Investigation of the mechanism of anti-PCNA aptamer-dependent inhibition of DNA replication revealed that the aptamer did not block formation, but was a component of PCNA/DNA polymerase δ or ϵ complexes. Additionally, the anti-PCNA aptamer competed with the primer-template DNA for binding to the PCNA/DNA polymerase δ or ϵ complex. Based on the observations, a model of anti-PCNA aptamer/PCNA complex-dependent inhibition of DNA replication was proposed.

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“…Selection of DNA aptamers was performed using the SELEX method as described previously ( 28 – 30 ) with minor modifications. Trimeric StrepTag-PCNA was used as the selection target.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex

Kowalska

Bartnicki

Fujisawa

et al. 2017

Nucleic Acids Research

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“…Affinity chromatography is currently mostly used by implying an affinity matrix, which can selectively bind to a recombinant fusion protein consisting of a fusion tag attached to the target protein [232][233][234]. An "Argi" system has been described in which the affinity matrix is a biotinylated DNA aptamer linked to streptavidine-agarose support [235]. The matrix could selectively bind fusion proteins with fusion tags of cationic CPPs consisting of either polyarginine or Tat49-57 peptide.…”

Section: Arginine In Ion-exchange Hydrophobic Interaction and Affinit...mentioning

confidence: 99%

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins

Gupta,

Uversky

2024

International Journal of Biological Macromolecules

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“…Arginine (R) is the most hydrophilic, basic natural amino acid residue with a pKa over 12, and possession of a positive charge within all biological environments. Because of these properties, synthetic polyarginine (poly-R) tags (i.e., a peptide sequence consisting of multiple, R repeats) serve a variety of experimental functions, including their use as a protein purification tag [42][43][44][45] , cellpenetrating peptide signal, multi-DNA-binding domain linker, and nucleolar trafficking signal 42,[46][47][48] . We hypothesized that a poly-R stretch coded in-between a NLS sequence and a downstream FP could act as an improved nuclear-localizing agent within neurons (Fig.…”

Section: Design Of a Nuclear-restricting Arginine-rich Nls (Arginls)mentioning

confidence: 99%

An arginine-rich nuclear localization signal (ArgiNLS) strategy for streamlined image segmentation of single-cells

Szelenyi,

Navarrete,

Murry

et al. 2023

Preprint

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High-throughput volumetric fluorescent microscopy pipelines can spatially integrate whole-brain structure and function at the foundational level of single-cells. However, conventional fluorescent protein (FP) modifications used to discriminate single-cells possess limited efficacy or are detrimental to cellular health. Here, we introduce a synthetic and non-deleterious nuclear localization signal (NLS) tag strategy, called ‘Arginine-rich NLS’ (ArgiNLS), that optimizes genetic labeling and downstream image segmentation of single-cells by restricting FP localization near-exclusively in the nucleus through a poly-arginine mechanism. A single N-terminal ArgiNLS tag provides modular nuclear restriction consistently across spectrally separate FP variants. ArgiNLS performance in vivo displays functional conservation across major cortical cell classes, and in response to both local and systemic brain wide AAV administration. Crucially, the high signal-to-noise ratio afforded by ArgiNLS enhances ML-automated segmentation of single-cells due to rapid classifier training and enrichment of labeled cell detection within 2D brain sections or 3D volumetric whole-brain image datasets, derived from both staining-amplified and native signal. This genetic strategy provides a simple and flexible basis for precise image segmentation of genetically labeled single-cells at scale and paired with behavioral procedures.Significance StatementQuantifying labeled cells in fluorescent microscopy is a fundamental aspect of modern biology. Critically, the use of short nuclear localization sequences (NLS) is a key genetic modification for discriminating single-cells labeled with fluorescent proteins (FPs). However, mainstay NLS approaches typically localize proteins to the nucleus with limited efficacy, while alternative non-NLS tag strategies can enhance efficacy at the cost of cellular health. Thus, quantitative cell counting using FP labels remains suboptimal or not compatible with health and behavior. Here, we present a novel genetic tagging strategy – named ArgiNLS – that flexibly and safely achieves FP nuclear restriction across the brain to facilitate machine learning-based segmentation of single-cells at scale, delivering a timely update to the behavioral neuroscientist’s toolkit.

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The Argi system: one-step purification of proteins tagged with arginine-rich cell-penetrating peptides

Cited by 8 publications

References 23 publications

Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex

Inhibition of DNA replication by an anti-PCNA aptamer/PCNA complex

Biological importance of arginine: A comprehensive review of the roles in structure, disorder, and functionality of peptides and proteins

An arginine-rich nuclear localization signal (ArgiNLS) strategy for streamlined image segmentation of single-cells

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