Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul 5;10(27):eadn9423.
doi: 10.1126/sciadv.adn9423. Epub 2024 Jul 5.

Piggybacking functionalized DNA nanostructures into live-cell nuclei

Golbarg M Roozbahani  1   2 , P L Colosi  3 , Attila Oravecz  4   5   6   7 , Elena M Sorokina  3 , Wolfgang Pfeifer  1   2 , Siamak Shokri  1 , Yin Wei  8 , Pascal Didier  7   9 , Marcello DeLuca  10 , Gaurav Arya  10 , László Tora  4   5   6   7 , Melike Lakadamyali  3   11   12 , Michael G Poirier  1   8   13 , Carlos E Castro  2   8
Affiliations

Piggybacking functionalized DNA nanostructures into live-cell nuclei

Golbarg M Roozbahani et al. Sci Adv. .

Abstract

DNA origami nanostructures (DOs) are promising tools for applications including drug delivery, biosensing, detecting biomolecules, and probing chromatin substructures. Targeting these nanodevices to mammalian cell nuclei could provide impactful approaches for probing, visualizing, and controlling biomolecular processes within live cells. We present an approach to deliver DOs into live-cell nuclei. We show that these DOs do not undergo detectable structural degradation in cell culture media or cell extracts for 24 hours. To deliver DOs into the nuclei of human U2OS cells, we conjugated 30-nanometer DO nanorods with an antibody raised against a nuclear factor, specifically the largest subunit of RNA polymerase II (Pol II). We find that DOs remain structurally intact in cells for 24 hours, including inside the nucleus. We demonstrate that electroporated anti-Pol II antibody-conjugated DOs are piggybacked into nuclei and exhibit subdiffusive motion inside the nucleus. Our results establish interfacing DOs with a nuclear factor as an effective method to deliver nanodevices into live-cell nuclei.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.. Concept for piggybacking DO nanostructures into the nucleus.
DO nanostructures functionalized with RNA Pol II–targeting antibodies and eight Cy5 fluorophores are electroporated into cells, bound to Pol II, and then are imported, or piggybacked, into the nucleus.
Fig. 2.
Fig. 2.. Fabrication and antibody labeling of DO nanostructures.
(A) Design schematic, oxDNA simulation, and TEM image of the 8HB DO structure. The simulation model depicts the base structure without the eight overhangs for fluorophore attachment. (B) Gel electrophoresis illustrates clear and efficient labeling of DO with one or two RNA Pol II antibodies (Abs) indicated by mobility shifts. TEM imaging confirmed efficient functionalization with one (C) or two (D) antibodies. Insets show a zoomed-in depiction of a single functionalized DO structure [compared to simulated 8HB structure with overhangs for fluorophore labels and antibody attached for size reference in (C)]. Scale bars, 30 nm.
Fig. 3.
Fig. 3.. Stability of DO nanostructures.
(A and B) Agarose gel electrophoresis (images show Cy5 fluorophore emission) revealed consistent mobility when 8HB structures were incubated at 37°C in (A) cell culture media or (B) cytoplasmic (C) or nuclear (N) extract from U2OS cells, verifying structural stability and fluorophore attachment integrity up to 24 hours. (C) TEM imaging also confirmed that structures remain intact, shown for the 24-hour nuclear extract condition (Scale bar, 30 nm). (D) Agarose gel electrophoresis also revealed no changes in mobility in electroporation buffer (R buffer) and after being subjected to electroporation for structures alone or for structures with one or two antibodies (Ab) attached. The ethidium bromide stain is shown on top, and Cy5 emission channel at the bottom. (E) TEM imaging also confirmed that structures remain intact and antibodies remain attached in R buffer and after electroporation. Insets show zoomed-in views of a single 8HB structure with one or two antibodies attached (Scale bars, 30 nm). (F and G) 8HB double-labeled with Cy3 and Cy5 electroporated into U2OS cells exhibit colocalization of both fluorophores in the cytoplasm (F) and the nucleus (G). Scale bars are indicated in the panels and insets.
Fig. 4.
Fig. 4.. Pol II antibodies facilitate the piggybacking of DOs to the nucleus.
(A) HILO imaging at the mid-plane of U2OS cells illustrates DO structures inside cells for 8HB-8Cy5 with zero antibodies (left, 8HB-8Cy5), one Pol II antibody (middle, anti–Pol II 8HB-8Cy5 1 Ab), or two Pol II antibodies (right, anti–Pol II 8HB-8Cy5 2 Ab). A clear increase in Cy5 fluorescence emission in cell nuclei is evident when 8HB-8Cy5 are labeled with one or two Pol II antibodies. Upper images show zoomed-in views of the nucleus and the cytoplasm for each condition. Scale bars, 10 μm. (B) The number of observed particles in the nuclei was quantified for each condition, showing that some 8HB-8Cy5 enter the nucleus even without antibodies, and there is a significant increase in nuclear localization with one or two antibodies on 8HB-8Cy5 structures. ****P ≤ 0.0001; ns, not significant.
Fig. 5.
Fig. 5.. Nuclear delivery of DOs in live cells.
(A) Representative images of live U2OS cells 24 hours after electroporation with nonfunctionalized 8HB DO structures (right, 8HB No Cy5) or 8HB-8Cy5 DO structures functionalized with either one anti–Pol II antibody (anti–Pol II 8HB-8Cy5; left), one anti-MBP antibody (anti-MBP 8HB-8Cy5; second from left), or 8HB-8Cy5 with no antibodies (8HB-8Cy5; second from right). White arrowheads point to nuclear particles representing single DO structures. Color bars indicate the fluorescence intensity range of the Cy5 signal (scale is ×1000). Nuclear Hoechst staining is shown in blue. Scale bars, 5 μm. (B) Combined violin, box, and jitter plots showing the quantification of nuclear particles. A significantly higher number of particles per nuclear area were detected in cells that were electroporated with anti–Pol II 8HB-8Cy5 compared to the other three conditions (H = 22.6447, P = 4.789 × 10−5, Kruskal-Wallis test). P values for pairwise comparisons using Dunn post hoc analyses are shown. n = 10. (C) Traces of tracked particles for anti–Pol II 8HB-8Cy5 within the nucleus depicted in (A). (D) Average MSD data measured from each of the eight nuclei analyzed and power-law fit over the entire data showing anomalous diffusion with an exponent of 0.4 (95% CI 0.2 to 0.6). (E) Average MSD data for Alexa 647–labeled anti–Pol II antibody–Pol II complex (anti–Pol II alone), without DO, from eight nuclei as in (D), showing anomalous diffusion with exponent 0.5 (95% CI 0.2 to 0.8).
See this image and copyright information in PMC

Update of

Similar articles

See all similar articles

References

    1. Yang D., Hartman M. R., Derrien T. L., Hamada S., An D., Yancey K. G., Cheng R., Ma M., Luo D., DNA materials: Bridging nanotechnology and biotechnology. Acc. Chem. Res. 47, 1902–1911 (2014). - PubMed
    1. He Z., Shi K., Li J., Chao J., Self-assembly of DNA origami for nanofabrication, biosensing, drug delivery, and computational storage. iScience 26, 106638 (2023). - PMC - PubMed
    1. Ma W., Zhan Y., Zhang Y., Mao C., Xie X., Lin Y., The biological applications of DNA nanomaterials: Current challenges and future directions. Signal Transduct. Target Ther. 6, 351 (2021). - PMC - PubMed
    1. Rothemund P. W. K., Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006). - PubMed
    1. Dey S., Fan C., Gothelf K. V., Li J., Lin C., Liu L., Liu N., Nijenhuis M. A. D., Saccà B., Simmel F. C., Yan H., Zhan P., DNA origami. Nat Rev Methods Primers 1, 13 (2021).
-