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Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins

Cassandra M. Stawicki, Torri E. Rinker, Markus Burns, Sonal S. Tonapi, Rachel P. Galimidi, Deepthi Anumala, Julia K. Robinson, Joshua S. Klein, Parag Mallick
doi: https://doi.org/10.1101/2021.07.30.454524
Cassandra M. Stawicki
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Torri E. Rinker
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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  • For correspondence: torrir@nautilus.bio
Markus Burns
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Sonal S. Tonapi
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Rachel P. Galimidi
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Deepthi Anumala
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Julia K. Robinson
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Joshua S. Klein
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Parag Mallick
1Nautilus Biotechnology, 201 Industrial Rd #310, San Carlos, CA 94070, USA
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Abstract

Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort cells. Fluorescent nanoparticle labels offer an excellent alternative to standard fluorescent labeling strategies due to their enhanced brightness, stability and multivalency; however, challenges in functionalization and characterization have impeded their use. This work introduces a straightforward approach for preparation of fluorescent nanoparticle probes using commercially available reagents and common laboratory equipment. Fluorescent polystyrene nanoparticles, Thermo Fisher FluoSpheres™, were used in proof-of-principle studies. Particle passivation was achieved by covalent attachment of amine-PEG-azide to carboxylated particles, neutralizing the surface charge from -47 to -17 mV. A conjugation-annealing handle and DNA aptamer probe was attached to the azide-PEG nanoparticle surface either through reaction of pre-annealed handle and probe or through a stepwise reaction of the nanoparticles with the handle followed by aptamer annealing. Nanoparticles functionalized with DNA aptamers targeting histidine tags and VEGF protein had high affinity (EC50s ranging from 2-7 nM) and specificity, and were more stable than conventional labels. This protocol for preparation of nanoparticle probes relies solely on commercially available reagents and common equipment, breaking down the barriers to use of nanoparticles in biological experiments.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted July 31, 2021.
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Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins
Cassandra M. Stawicki, Torri E. Rinker, Markus Burns, Sonal S. Tonapi, Rachel P. Galimidi, Deepthi Anumala, Julia K. Robinson, Joshua S. Klein, Parag Mallick
bioRxiv 2021.07.30.454524; doi: https://doi.org/10.1101/2021.07.30.454524
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Modular Fluorescent Nanoparticle DNA Probes for Detection of Peptides and Proteins
Cassandra M. Stawicki, Torri E. Rinker, Markus Burns, Sonal S. Tonapi, Rachel P. Galimidi, Deepthi Anumala, Julia K. Robinson, Joshua S. Klein, Parag Mallick
bioRxiv 2021.07.30.454524; doi: https://doi.org/10.1101/2021.07.30.454524

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