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Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine

Nature Protocols volume 6, pages 2022–2034 (2011)Cite this article

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Abstract

Individual genes can be targeted with siRNAs. The use of nucleic acid nanoparticles (NPs) is a convenient method for delivering combinations of specific siRNAs in an organized and programmable manner. We present three assembly protocols to produce two different types of RNA self-assembling functional NPs using processes that are fully automatable. These NPs are engineered based on two complementary nanoscaffold designs (nanoring and nanocube), which serve as carriers of multiple siRNAs. The NPs are functionalized by the extension of up to six scaffold strands with siRNA duplexes. The assembly protocols yield functionalized RNA NPs, and we show that they interact in vitro with human recombinant Dicer to produce siRNAs. Our design strategies allow for fast, economical and easily controlled production of endotoxin-free therapeutic RNA NPs that are suitable for preclinical development.

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Figure 1: Schematic representation of three different cassette-based assembly protocols for siRNA-functionalized NPs.
Figure 2: Quality control experiments for assembled RNA NPs.
Figure 3: Functional control experiments for assembled RNA NPs.

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Acknowledgements

This research was supported (in part) by the Intramural Research Program of the US National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research. This work has been funded in whole or in part with federal funds from the National Cancer Institute, NIH, under contract no. HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US government. This research was also supported by NIH grant no. R01GM-079604 (to L.J.). We are grateful to B. Neun for technical assistance with the LAL assay and to J. Hall for help with manuscript preparation.

Author information

Author notes

  1. Kirill A Afonin and Wade W Grabow: These authors contributed equally to this project.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Biomolecular Science and Engineering Program, University of California, Santa Barbara, California, USA

    Kirill A Afonin, Wade W Grabow, Faye M Walker & Luc Jaeger

  2. Center for Cancer Research Nanobiology Program, National Cancer Institute (NCI)-Frederick, Frederick, Maryland, USA

    Kirill A Afonin & Bruce A Shapiro

  3. Basic Science Program, Science Applications International Corporation (SAIC)-Frederick, Inc., NCI-Frederick, Frederick, Maryland, USA

    Eckart Bindewald

  4. Nanotechnology Characterization Laboratory, Advanced Technology Program, SAIC-Frederick Inc., NCI-Frederick, Frederick, Maryland, USA

    Marina A Dobrovolskaia

Authors
  1. Kirill A Afonin
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  2. Wade W Grabow
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  5. Marina A Dobrovolskaia
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  6. Bruce A Shapiro
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  7. Luc Jaeger
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Contributions

K.A.A., W.W.G., B.A.S. and L.J. conceived and designed the experiments. K.A.A., W.W.G., E.B., B.A.S. and L.J. contributed to the sequence and 3D model design. K.A.A., W.W.G. and F.M.W. performed self-assembly PAGE and dicing experiments. K.A.A. and F.M.W. performed DLS experiments. K.A.A., W.W.G., B.A.S. and L.J. analyzed the data. M.A.D. and K.A.A performed the LAL assay. K.A.A., W.W.G., B.A.S. and L.J. co-wrote the paper.

Corresponding authors

Correspondence to Bruce A Shapiro or Luc Jaeger.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Data 1

RNA sequences used in this project. (DOCX 26 kb)

Supplementary Fig. 1

Schematic demonstration of 3′ ring scaffold functionalization with siRNA duplex. Please note that depending on tasks siRNA sense and antisense strands can be swapped. (EPS 509 kb)

Supplementary Fig. 2

Native PAGE results for native PAGE of assembly experiments for different protocols of duplex formation (between concatenated with sense strand cube strand D (siD) and corresponding radiolabeled antisense (Ant*)). (EPS 941 kb)

Supplementary Fig. 3

Native PAGE results for one-pot assembly experiments for the assembly of the cubes 3′ side concatenated with up to six identical siRNAs (eGFPS1) with corresponding yields of assembly. Radiolabeled strands are indicated with asterisks. Please note that depending on number and orientation of siRNA concatenated scaffold strands the shapes of NPs and their relative gel shifts may be slightly different (cubes functionalized with 4 siRNA duplexes). (EPS 5818 kb)

Supplementary Fig. 4

Native PAGE results for one-pot, step-wise and duplex assemblies of cubes and rings 3′ side concatenated with six different siRNAs (HIV-1). As the controls, assembled cubes and rings functionalized with 6 identical siRNAs (Figure 2) are used. (EPS 3059 kb)

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Afonin, K., Grabow, W., Walker, F. et al. Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nat Protoc 6, 2022–2034 (2011). https://doi.org/10.1038/nprot.2011.418

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