Bioinspired designer DNA NanoGripper for virus sensing and potential inhibition

Abstract

DNA has shown great biocompatibility, programmable mechanical properties, and precise structural addressability at the nanometer scale, rendering it a material for constructing versatile nanorobots for biomedical applications. Here, we present the design principle, synthesis, and characterization of a DNA nanorobotic hand, called DNA NanoGripper, that contains a palm and four bendable fingers as inspired by naturally evolved human hands, bird claws, and bacteriophages. Each NanoGripper finger consists of three phalanges connected by three rotatable joints that are bendable in response to the binding of other entities. NanoGripper functions are enabled and driven by the interactions between moieties attached to the fingers and their binding partners. We demonstrate that the NanoGripper can be engineered to effectively interact with and capture nanometer-scale objects, including gold nanoparticles, gold NanoUrchins, and SARS-CoV-2 virions. With multiple DNA aptamer nanoswitches programmed to generate a fluorescent signal that is enhanced on a photonic crystal platform, the NanoGripper functions as a highly sensitive biosensor that selectively detects intact SARS-CoV-2 virions in human saliva with a limit of detection of ~100 copies per milliliter, providing a sensitivity equal to that of reverse transcription quantitative polymerase chain reaction (RT-qPCR). Quantified by flow cytometry assays, we demonstrated that the NanoGripper-aptamer complex can effectively block viral entry into the host cells, suggesting its potential for inhibiting virus infections. The design, synthesis, and characterization of a sophisticated nanomachine that can be tailored for specific applications highlight a promising pathway toward feasible and efficient solutions to the detection and potential inhibition of virus infections.

Fig. 1.. Schematic of DNA NG design, functionalization, and utility in viral detection and inhibition.

(A) Configuration and dimension of the DNA NG that contains a central palm and four bendable fingers, where F, Z, and R represent the finger, rotation axis and rotation joint, respectively. (B) Functionalization of the DNA NG for interacting with 3D nano-objects, including AuNP (~50 and 100 nm), AuNUP (~80 nm), and SARS-CoV-2 (~100 nm).“d” indicates diameter. (C) Schematic drawing of SARS-CoV-2 detection and cell entry inhibition using DNA NG.

Fig. 2.. DNA NG design.

(A) Determination of optimized finger and palm cross section sizes. (B) Schematic of the favorite finger-palm connection design that offers an outward flip of the fingers to ensure that the NG fingers stay in open configurations in free solution (i) and schematic of scaffold DNA routing displayed using MagicDNA (ii) and oxView (iii).

Fig. 3.. Synthesis and characterization of DNA NG.

(A) Schematic of DNA NG assembly using programmed thermal annealing. (B) AGE shows the NG formation (as a predominate band) in the buffer with different Mg²⁺ concentrations. All NG samples were purified to remove excess staple strands before subsequent characterization and assays. (C) Sample microscopy images of the DNA NG. See more AFM, TEM, and cryo-EM images of DNA NG with lower magnification in the Supplementary Materials. (i) AFM image. (ii) TEM image after negative staining. (iii) Cryo-EM image. (D) Side-by-side comparison of DNA NG structure obtained from TEM imaging with the corresponding NG 3D model configuration. Scale bars indicate 100 nm.

Fig. 4.. Capture of AuNPs and SARS-CoV-2 by DNA NG.

(A) AFM and TEM images show AuNPs’ interaction with DNA NG carrying ssDNA with a sequence complementary to the ssDNA coated on AuNPs (50 or 100 nm). White and blue arrows indicate the NG (finger) and AuNP, respectively. Scale bars indicate 50 nm. (B) Cryo-EM images show the capture of SARS-CoV-2 by the DNA NG carrying multiple spike protein–targeting aptamers. Red arrows indicate the NG fingers. Scale bars indicate 50 nm.

Fig. 5.. Capture and count of AuNUPs on a PC surface by DNA NG.

(A) Schematic of the capture and counting of NG-captured AuNUPs. In the zoom in subpanel, AuNUPs could be captured by NGs and then brought to the surface through the hybridization of the “Anchor” ssDNAs on the palm (indicated by red arrows) and complementary surface “capture” DNA (indicated by a white arrow) on the PC surface. NG interacts with AuNUPs via biotin-streptavidin binding. Size and shape of the AuNUPs were characterized using SEM and TEM imaging (fig. S26). (B) PRAM images of surface-captured AuNUPs (black dots) by DNA NGs tested at different particle concentrations and different incubation time points. Free biotinylated ssDNA (not attached to NG) was used as a negative control probe to show the background of the PC surface scan by PRAM. (C) Transmission scan of the PC surface in the presence or absence of surface-immobilized NGs. (D) Plots for the counts of surface-captured AuNUPs by DNA NGs tested at different concentrations and different incubation time points. Data are presented as mean ± SD; n = 9 replicates.

Fig. 6.. PC-enhanced detection of SARS-CoV-2 by a DNA NG sensor.

(A) Schematic of viral capture and fluorescent signal generation mechanism. (B) Schematic of PC-NG hybrid system shows that the NG aptamer can capture SARS-CoV-2 in free solution and then bring it to the PC surface via anchor (on NGs) and tether DNA (on PCs) hybridization. (C) PC near-field enhancement and PCEF scanning microscope experimentally obtained with nearly 250-fold signal enhancement compared with an NG reporter on a glass substrate. (D) PC-enhanced digital counting and dose response of SARS-CoV-2 detection. Representative scanning images of PC surface with captured SARS-CoV-2 at various viral concentrations (10² to 10⁷ copies/ml) after a 10-min incubation with the NG sensor. Scale bars, 50 μm. (E) Quantification of captured SARS-CoV-2 virions on the PC surface at different viral concentrations. The red curve is a result of connecting the points representing the average values of the fluorescence signal counts at different virus concentrations. Assays were performed in triplicate with a similar observation. Blue baseline represents the negative controls containing only buffer solution. Error bars represent the SDs of three biologically independent measurements.

Fig. 7.. Flow cytometry assay showing that the DNA NG–aptamer complex can block virus-cell interaction and virus entry with very high efficacy.

(A) Comparative flow cytometry analysis of concentration-matched free aptamers and DNA NG–aptamer complex–mediated virus inhibition demonstrates highly effective prevention of viral entry into cells. (B) Viral entry into cell quantitated using intracellular staining of SARS-CoV-2 spike RBD with FITC-conjugated mAbs further demonstrates the antiviral efficacy of DNA NG–aptamer constructs. Five biological replicates were included for each condition. Statistical significance is indicated by P values (<0.005) as determined with unpaired two-tailed Student’s t test. The untreated virus serves as a positive infection control (no aptamer or NG-aptamer complex added). Uninfected cell represents a negative infection control (no virus added).

Fig. 8.. SARS-CoV-2 viral cell entry inhibition by DNA NG.

SARS-CoV-2 virions (red dots), VeroE6 cell membrane structures (yellow), and the cell nuclei (blue) were labeled with FAM, Dil, and Hoescht stains, respectively. Representative SARS-CoV-2 viral particles are indicated by white arrows. (A) SARS-CoV-2 virions accumulate in the cells over time in the presence of monomeric aptamers. A SARS-CoV-2 spike–targeting, FAM fluorophore–labeled aptamer was used as a moiety to tag the virus particles for fluorescence imaging; it also served as an aptamer-only control to provide visualization-based qualitative analysis and comparison of its antiviral function with the DNA NG aptamer–based viral inhibitor. (B) SARS-CoV-2 viral entry is inhibited over time by DNA NG treatment. Each panel is accompanied by three cross sections of the main view (along the dotted lines in the main images). Confocal images show that free aptamer–bound SARS-COV-2 particles accumulate in the cell (A), and DNA NG–aptamer–bound SARS-CoV-2 particles are largely prevented from cell entry (B). Assays were performed in triplicate with similar observations. Scale bars, 10 μm.