Cellular Uptake of Tile-Assembled DNA Nanotubes

DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.

[1]  P. Swaan,et al.  Endocytic mechanisms for targeted drug delivery. , 2007, Advanced drug delivery reviews.

[2]  Antti-Pekka Eskelinen,et al.  Virus-encapsulated DNA origami nanostructures for cellular delivery. , 2014, Nano letters.

[3]  Andrew J Turberfield,et al.  Single-molecule protein encapsulation in a rigid DNA cage. , 2006, Angewandte Chemie.

[4]  Hendrik Dietz,et al.  Magnesium-free self-assembly of multi-layer DNA objects , 2012, Nature Communications.

[5]  D. Frenkel,et al.  Effect of inert tails on the thermodynamics of DNA hybridization. , 2014, Journal of the American Chemical Society.

[6]  P. Yin,et al.  Complex shapes self-assembled from single-stranded DNA tiles , 2012, Nature.

[7]  C. Berking,et al.  Proapoptotic signaling induced by RIG-I and MDA-5 results in type I interferon-independent apoptosis in human melanoma cells. , 2009, The Journal of clinical investigation.

[8]  C. Klein,et al.  Selective bispecific T cell recruiting antibody and antitumor activity of adoptive T cell transfer. , 2015, Journal of the National Cancer Institute.

[9]  William M. Shih,et al.  Addressing the Instability of DNA Nanostructures in Tissue Culture , 2014, ACS nano.

[10]  J. Rossi,et al.  Therapeutic Potential of Aptamer-siRNA Conjugates for Treatment of HIV-1 , 2012, BioDrugs.

[11]  T. Blankenstein,et al.  Retroviral vectors for high-level transgene expression in T lymphocytes. , 2003, Human gene therapy.

[12]  Andrew D. Miller Cationic Liposomes for Gene Therapy , 1998 .

[13]  Shawn M. Douglas,et al.  Self-assembly of DNA into nanoscale three-dimensional shapes , 2009, Nature.

[14]  K. Kataoka,et al.  Block copolymer micelles for drug delivery: design, characterization and biological significance. , 2001, Advanced drug delivery reviews.

[15]  Wael Mamdouh,et al.  Single-molecule chemical reactions on DNA origami. , 2010, Nature nanotechnology.

[16]  P. Rothemund Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[17]  Björn Högberg,et al.  DNA origami delivery system for cancer therapy with tunable release properties. , 2012, ACS nano.

[18]  Daniel G. Anderson,et al.  Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo siRNA Delivery , 2012, Nature nanotechnology.

[19]  Luvena L. Ong,et al.  Three-Dimensional Structures Self-Assembled from DNA Bricks , 2012, Science.

[20]  T. Liedl,et al.  Nucleic acid nanostructures for biomedical applications. , 2013, Nanomedicine.

[21]  Shawn M. Douglas,et al.  A Logic-Gated Nanorobot for Targeted Transport of Molecular Payloads , 2012, Science.

[22]  J. Kjems,et al.  Quantification of cellular uptake of DNA nanostructures by qPCR. , 2014, Methods.

[23]  Tim Liedl,et al.  Nanoscale structure and microscale stiffness of DNA nanotubes. , 2013, ACS nano.

[24]  Chengde Mao,et al.  DNA nanotubes as combinatorial vehicles for cellular delivery. , 2008, Biomacromolecules.

[25]  H. Pei,et al.  Self-assembled multivalent DNA nanostructures for noninvasive intracellular delivery of immunostimulatory CpG oligonucleotides. , 2011, ACS nano.

[26]  Hyukjin Lee,et al.  Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates. , 2014, Chemical communications.

[27]  Harry M. T. Choi,et al.  Programming DNA Tube Circumferences , 2008, Science.

[28]  Tim Liedl,et al.  Cellular immunostimulation by CpG-sequence-coated DNA origami structures. , 2011, ACS nano.

[29]  이 익모,et al.  Nanomaterials , 2021, Bionanotechnology.

[30]  N. Seeman Construction of three-dimensional stick figures from branched DNA. , 1991, DNA and cell biology.

[31]  G. Hannon,et al.  Small RNA sorting: matchmaking for Argonautes , 2011, Nature Reviews Genetics.

[32]  Hao Yan,et al.  DNA origami as a carrier for circumvention of drug resistance. , 2012, Journal of the American Chemical Society.

[33]  William M. Shih,et al.  Virus-Inspired Membrane Encapsulation of DNA Nanostructures To Achieve In Vivo Stability , 2014, ACS nano.

[34]  William M. Shih,et al.  A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron , 2004, Nature.