Results
1. LSM: Internalization Validation
In our design, DNA origami will first bind to the target bacteria through the aptamers' targeting effect, while the G4/hemin DNAzymes will catalyze H2O2 produced by the wound site, generating reactive oxygen species (ROS) to permeabilize the bacterial membrane. Subsequently, the DNA origami will enter the cell through passive diffusion, which is known as internalization. The effect of membrane-permeabilization and targeting had already been examined by previous experiments. The internalization validation assay aimed to systematically assess these abilities and complete the final verification of the DNA origami entry into target cells, qualitatively and quantitatively.
To intuitively visualize the internalization of DNA origami, we continued to employ confocal laser scanning microscopy (LSM) for validation. In this experiment, the membrane-permeabilization ability of DNA origami was necessary. Therefore, G4/hemin DNAzymes (GH) were loaded onto the DNA origami in this experiment. To visualize the DNA origami, Cy5 was used as a fluorescent label, which could be detected under a fluorescence microscope. Two groups were designed to compare the internalization ability: Cy5-labeled DOPAMGH as the control group and Cy5-labeled DOAPAMGH as the experimental group. After assembly and incubation with E. coli for 15min at 37°C, the mixture was washed with PBS three times and observed under LSM 880 (Zeiss, Germany). The signals without co-localization with the bacteria were regarded as uncleaned Cy5 or Cy5-labeled DNA origami, which were subtracted as background, while the signals that were co-localized with bacteria were regarded as the DNA origami that had effectively entered the cell, which were selected and conducted mean fluorescence intensity (MFI) quantitative analysis. Detailed information can be found in the following protocol (12 Internalization validation by LSM) and handbook (Zeiss Confocal Microscope - LSM880 Handbook).
Under the LSM microscope, the Cy5-labeled DOAPAMGH was obviously observed to indicate a significantly greater internalization efficiency (Figure 1). MFI analysis further revealed that the MFI of the Cy5-labeled DOAPAMGH group was significantly higher than that of the Cy5-labeled DOPAMGH group (Figure 2). These results showed that, with the addition of the aptamer, the ability of internalization increased, in line with expectations.
Figure 1. Internalization of Cy5-labeled DOPAMGH and DOAPAMGH on E. coli. With the addition of the aptamer, the amount of fluorescence entering the cells increased, and the area with fluorescence also expanded. Scale bar: 20µm × 20µm.
Figure 2. Quantification of the mean fluorescence intensity (MFI) of E. coli cells after the incubation of Cy5-labeled DOPAMGH with or without the modification of the aptamer. - Aptamer: Cy5-labeled DOPAMGH. + Aptamer: Cy5-labeled DOAPAMGH. Data represent the mean ± s.d.. Statistical significance was calculated by the unpaired two-tailed Student's t-test, with * representing P < 0.05.
2. Blue/White Selection: Overall Validation
In the above experiments, we have validated the loading of sgRNAL/Cas9 complex, the loading of G4/hemin DNAzymes, the membrane-permeabilization ability, the targeting ability, and the internalization. The eventual objective is to confirm the overall function of our DNA origami-based gene editing system.
To validate the efficacy of our origami-CRISPR/Cas9 platform, we assembled a fully functional DNA origami (DOAPAMRCGH) with aptamer, sgRNAL/Cas9 complex, and G4/hemin DNAzymes. We applied it to E. coli MG1655 carrying the lacZ gene. lacZ encodes a beta-galactosidase that transforms the white X-gal into galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter product then undergoes spontaneous dimerization and oxidation to form a blue-colored indigo pigment (1). Therefore, if our DNA origami successfully targets and inactivates the lacZ gene in E. coli MG1655, the colony cultured on LB plates supplemented with X-gal and Isopropyl β-D-1-thiogalactopyranoside (IPTG) will appear white rather than blue. We designed two groups: E. coli MG1655 treated by buffer as the control group, and E. coli MG1655 treated by DOAPAMRCGH as the experimental group. After incubation and plating the mixture, the functionality of our DNA origami-based gene editing system could be assessed by the color of the colonies. Details of the Blue/White Selection are provided in the protocol (13 Blue_white selection).
Numerous blue colonies but no white colonies were observed in the control group after incubation (Figure 3A), whereas the experiment group treated with DOAPAMRCGH displayed both blue and white colonies (Figure 3B), indicating that DOAPAMRCGH successfully inactivated the lacZ gene in a portion of the bacterial population. The result of the blue/white selection confirmed the overall function of our DNA origami-based gene editing system.
Figure 3. Blue/White selection result. A. Numerous blue colonies were observed in the control group. B. Both blue and white colonies were observed in the experiment group treated with the DOAPAMRCGH.
Protocol
12. Internalization validation by LSM (click me to open the protocol!)
12.2 Handbook: LSM (click me to open the handbook!)
13. Blue/white selection (click me to open the protocol!)
Notebook
Notebook for Functional Validation (click me to open the Notebook!)
Reference
- Julin D. Blue-White Selection. In: Molecular Life Sciences [Internet]. Springer, New York, NY; 2014 [cited 2025 Sep 29]. p. 1–2. Available from: https://link.springer.com/rwe/10.1007/978-1-4614-6436-5_94-2
