Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity

Abstract

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

Figure 1:. The construction and membrane-insertion activities of PLY-DNA rings.

(A) Schematical illustration of PLY-anti-handle conjugation (left) and SDS-PAGE assay (right) showing the gel mobility shift of PLY before and after ssDNA anti-handle conjugation. (B) Cartoon models showing the dimensions and handle positions of the DNA-origami ring (left) and a negative-stain TEM micrograph of the purified DNA rings. Three sets of handles with orthogonal sequences (blue, green, and red curls) were strategically placed for attaching fluorophores and proteins via handle/anti-handle hybridization. Scale bar: 50 nm. (C) Schematics and negative-stain EM micrographs showing the assembly of PLY-modified DNA-origami rings and their membrane insertion. Scale bars: 50 nm. (D) A cryo-EM image showing a liposome perforated by a DNA-origami-corralled PLY pore. Scale bar: 50 nm. (E) Sheep erythrocytes lysis assay comparing membrane puncturing by unconjugated PLY and PLY-DNA rings. DNA-origami rings without PLY modification serve as a negative control. Data points are shown with mean and standard deviation from three independent trials.

Figure 2:. PLY-DNA-origami produces transmembrane nanopores that facilitate the diffusion of macromolecules into GUVs.

Shown from left to right are the schematics and representative images of GUVs 20 minutes after adding buffer only, anti-handle-conjugated PLY, PLY-DNA rings, and DNA-origami rings without PLY. Pseudo-colors: Cyan = Cy3 (DNA ring), red = Cy5 (GUV), and green = FITC (dextran). Scale bars: 5 μm. Bar graphs at the bottom denote the percentages of GUVs with observable dextran influx.

Figure 3:. Diffusion of macromolecules across PLY-DNA-origami pores is size-dependent.

(A) Schematics of the FRAP assay on a GUV with PLY-DNA nanopores. After the interior of the GUV is equilibrated with the exterior containing FITC-dextran, the interior is photobleached and monitored for the fluorescence signal recovery caused by the exchange of FITC-dextran through the nanopores. (B) Representative FRAP traces of perforated GUVs mixed with FITC-dextran of various sizes. The slope K (red line) denotes the initial rate of FITC fluorescence recovery after photobleaching (time 0). Insets: Confocal micrographs of GUVs at selected time points during the FRAP experiment. Scale bars: 5 μm. (C) The initial recovery rates (K) normalized by the radii of GUVs are used to compare the diffusion rates of dextrans of various sizes through PLY-DNA pores. Data points are shown with mean and standard deviation. ****: P < 0.0001, two-tailed Mann–Whitney tests, U = 17 (left) and 9 (right). See Supporting Information for details of statistical analyses.

Figure 4:. Incorporating Nsp1 into PLY-DNA rings alters the nanopore size selectivity.

(A) A schematic illustrating a PLY-DNA nanopore modified by 36 copies of Nsp1 grafted to the DNA central channel. (B) An SDS-agarose gel showing the empty DNA ring, PLY-DNA ring, and Nsp1-modified PLY-DNA ring structures. (C) Negative-stain EM micrographs of PLY-DNA rings modified by Nsp1. Scale bar: 50 nm. (D) Schematics (left) and representative fluorescence confocal images showing the influx of FITC-dextran (green) through PLY-DNA pores (top) and Nsp1-modified PLY-DNA pores (bottom) into Cy5-labeled GUVs (red) after 15 min co-incubation. Scale bars: 5 μm. (E) Representative FRAP traces of GUVs containing Nsp1-modified PLY-DNA nanopores mixed with 20kDa (top) and 40kDa (bottom) FITC-dextran. K (red line) denotes the initial rate of FITC fluorescence recovery after photobleaching (time 0). Insets: Confocal micrographs of GUVs at selected time points during the FRAP experiment. Scale bars: 5 μm. (F) The initial rates of fluorescence recovery (K) normalized by the radii of GUVs are used to compare the diffusion rates of 20 kDa and 40 kDa dextrans through empty (grey, same as those shown in Figure 3C) and Nsp1-modified (blue) PLY-DNA pores. Data points are shown with mean and standard deviation. ****: P < 0.0001, two-tailed Mann–Whitney test, U = 17 (left), 27 (middle), and 34 (right). See Supporting Information for details of statistical analyses.