Active Nuclear Import of Mammalian Cell-Expressible DNA Origami

DNA origami enables the creation of complex 3D shapes from genetic material. Future uses could include the delivery of genetic instructions to cells, but nuclear import remains a major barrier to gene delivery due to the impermeability of the nuclear membrane. Here we realize active nuclear import of DNA origami objects in dividing and chemically arrested mammalian cells. We developed a custom DNA origami single-strand scaffold featuring a mammalian-cell expressible reporter gene (mCherry) and multiple Simian virus 40 (SV40) derived DNA nuclear targeting sequences (DTS). Inclusion of the DTS within DNA origami rescued gene expression in arrested cells, indicating that active transport into the nucleus occurs. Our work successfully adapts mechanisms known from viruses to promote the cellular expression of genetic instructions encoded within DNA origami objects.


Materials & Methods
Plasmid cloning. Plasmids encoding for custom scaffolds were created via standard cloning techniques. All plasmids were created via Golden gate assembly using BsaI-HF®v2 (NEB cat. no. R3733), together with T4 DNA ligase (NEB cat. no. M0202). For each plasmid, appropriate cut sites were introduced with PCR (Q5® High-Fidelity 2x Master Mix, NEB, as per manufacturer's protocol), and the assembly was conducted as per manufacturer's protocol. Plasmids were verified using restriction digests and DNA sequencing (Eurofins genomics, Ebersberg Germany).
Touchdown PCR with primers for all constructs are detailed in Table S1. In all cases, PCR products were confirmed by agarose gel electrophoresis (AGE), bands were excised, and fragments were extracted (Qiagen QIAquick Gel Extraction Kit) as per manufacturer's protocol. For the introduction of DTS sequences, custom sequences encoding for the 72 bp SV40 DTS and BsaI cleavage sites were ordered as ssDNA from IDT and were annealed in-house directly prior to the Golden gate assembly reaction. REV GTACACGGTCTCGagtcgaggctgatcagcgg *Sequences were annealed and used directly in the Golden gate assembly reaction, as opposed to being used as primers.
Scaffold production. The custom scaffolds were produced based on the plasmids described above.
A protocol to produce custom scaffolds was presented previously 1,2 and is described in detail in the following section.
For ssDNA production, chemically competent DH5α E. coli cells were cotransformed with both the scaffold plasmid of interest, and a helper plasmid (Addgene, # 120346). Transformations were plated on agar plates containing 100 μg/mL carbenicillin and 50 μg/mL kanamycin. After growing overnight, a single colony was picked and grown in a 5 mL pre-culture (2xYT medium, 100 μg/mL carbenicillin, 50 μg/mL kanamycin) at 37 °C with shaking. After turning turbid (~10 h), the pre-cultures were transferred to 2.5 L Ultra Yield flasks (Thomson) containing 750 mL of 2xYT medium (50 μg/mL carbenicillin, 30 μg/mL kanamycin, 5 mM MgCl2), and were grown overnight at 37 °C. 50 μL anti-foam (A8311, Sigma-Aldrich) was added per flask to avoid foam formation. The next day, the culture was transferred to 750 mL centrifuge bottles and the bacteria were removed by centrifugation at 4700g for 45 min at 4 °C. The supernatants were transferred to fresh centrifuge bottles where 30 g/L polyethylene glycol 8000 (PEG-8000) and 30 g/L NaCl was added and mixed for 30 min, r.t. to precipitate the phages. Afterwards, the phages were pelleted by centrifugation (1 h, 4700g, 4 °C), and the pellet was resuspended in 4 mL 1×TE buffer. Samples were transferred to 50 mL falcon tubes, and residual bacteria was pelleted by centrifugation (20 min, 15 000g, 4 °C). The supernatant was transferred to a fresh falcon tube and the single stranded DNA was extracted from the phages. First, 10 mL lysis buffer (Qiagen P2, cat. no. 19052) was added and mixed gently by inversion. Afterwards, 7.5 mL neutralization buffer (Qiagen P3, cat. no. 19053) was added and mixed by inversion. Samples were incubated on ice for 15 min, and then centrifuged (25 min, 16 000g, 4 °C). The supernatant was transferred to fresh falcon tubes and ethanol (22.5 mL/tube, 4 °C) was added. The falcon was incubated in an ice water bath for 30 min and centrifuged (20 min, 16 000g, 4 °C) to collect precipitated ssDNA. The ssDNA pellet was then washed with an additional 10 mL 75% ethanol, incubated (10 min, ice water bath), and centrifuged (20 min, 16 000g, 4 °C). The supernatant was carefully removed, and residual ethanol was evaporated, before the pellet was dissolved in 1-2 mL 1×TE. Scaffold concentrations were measured with a NanoDrop™8000 Spectrophotometer (Thermo Scientific) using the absorbance at 260 nm. The size and sequence of the scaffold was verified using agarose gel electrophoresis and sequencing (Eurofins genomics).