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Protein transfection via spherical nucleic acids

Nature Protocols volume 17, pages 327–357 (2022)Cite this article

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Abstract

The efficient transfection of functional proteins into cells can serve as a means for regulating cellular processes toward solving fundamental challenges in biology and medicine. However, the use of proteins as effective intracellular agents is hindered by their low cellular uptake and susceptibility to degradation. Over the past 15 years, our group has been developing spherical nucleic acids (SNAs), nanoparticles functionalized with a dense radially oriented shell of nucleic acids. These structures actively enter cells and have opened new frontiers in chemical sensing, biodiagnostics and therapeutics. Recently, we have shown that proteins can be used as structurally precise and homogeneous nanoparticle cores in SNAs. The resultant protein SNAs (ProSNAs) allow previously cell-impermeable proteins to actively enter cells, exhibit high degrees of stability and activity both in cell culture and in vivo, and show enhanced pharmacokinetics. Consequently, these modular structures constitute a plug-and-play platform in which the protein core and nucleic acid shell can be independently varied to achieve a desired function. Here, we describe the synthesis of ProSNAs through the chemical modification of solvent-accessible surface residues (3–5 d). We also discuss design considerations, strategies for characterization, and applications of ProSNAs in cellular transfection, biological sensing and functional enzyme delivery in vivo.

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Fig. 1: Synthesis, purification and characterization of ProSNAs.
Fig. 2: Two-step assay for glucose detection.
Fig. 3: Working principle for ONPG-based β-Gal activity assay.
Fig. 4: Working principle of X-Gal assay for measuring β-Gal activity.
Fig. 5: Working principle of fluorescence-based assay for measuring β-Gal activity.
Fig. 6: Characterization of ProSNAs.
Fig. 7: Uptake and activity of β-Gal SNAs in cell culture.
Fig. 8: Validation of a GOx SNA-based two-step assay for detection of glucose.
Fig. 9: Biodistribution and activity of ProSNAs in vivo.

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Data availability

The main data discussed in this protocol are available in the supporting primary research papers (https://doi.org/10.1021/jacs.0c06866, https://doi.org/10.1021/jacs.5b09711, and https://doi.org/10.1021/acscentsci.0c00313). Raw data for the figures shown can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

This material is based on research sponsored by Air Force Office of Scientific Research award FA9550-17-1-0348 and Air Force Research Laboratory agreement FA8650-15-2-5518. The U.S. Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright notation thereon. The views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of Air Force Research Laboratory or the U.S. Government. S.B.E. was supported in part by the Chicago Cancer Baseball Charities and the H Foundation at the Lurie Cancer Center of Northwestern University. We acknowledge all former group members who contributed to the development of the methods described in this protocol.

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  1. These authors contributed equally: Sasha B. Ebrahimi, Devleena Samanta.

Authors and Affiliations

  1. Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA

    Sasha B. Ebrahimi & Chad A. Mirkin

  2. International Institute for Nanotechnology, Northwestern University, Evanston, IL, USA

    Sasha B. Ebrahimi, Devleena Samanta, Caroline D. Kusmierz & Chad A. Mirkin

  3. Department of Chemistry, Northwestern University, Evanston, IL, USA

    Devleena Samanta, Caroline D. Kusmierz & Chad A. Mirkin

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Contributions

S.B.E. and D.S. contributed equally. All authors contributed to the development of the protocol and the design of the experiments. All authors wrote and approved the manuscript.

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Correspondence to Chad A. Mirkin.

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Key references using this protocol:

Brodin, J. D. et al. J. Am. Chem. Soc. 137, 14838–14841 (2015): https://doi.org/10.1021/jacs.5b09711

Kusmierz, C. D. et al. ACS Cent. Sci. 6, 815–822 (2020): https://doi.org/10.1021/acscentsci.0c00313

Samanta, D. et al. J. Am. Chem. Soc. 142, 13350–13355 (2020): https://doi.org/10.1021/jacs.0c06866

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Ebrahimi, S.B., Samanta, D., Kusmierz, C.D. et al. Protein transfection via spherical nucleic acids. Nat Protoc 17, 327–357 (2022). https://doi.org/10.1038/s41596-021-00642-x

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