(W0930-04-23) Three Dimensional Industrial DNA Nanorobot as a Functional Drug Delivery System

Purpose: Nanoscale industrial robots have potential as manufacturing platforms, capable of automatically performing repetitive tasks to handle and produce nanomaterials with consistent precision and accuracy. We demonstrate a DNA industrial nanorobot, that fabricates a three-dimensional (3D) optically active, chiral structure from optically inactive parts. By making use of externally controlled temperature and ultraviolet (UV) light, our programmable robot, ~ 100 nanometers in size, grabs different parts, positions and aligns them so that they can be "welded", releases the construct and returns to its original configuration ready for its next operation. Our introduction of multiple-axis precise folding and positioning as a tool/technology for nano manufacturing will open the door to more complex and useful nano-micro devices as novel drug delivery systems.

Methods: Materials: Five-nanometer AuNPs in cetyltrimethylammonium bromide were purchased from Ted Pella Inc. Twenty-nanometer (20 nm by 5 nm by 5 nm) and 25-nm (25 nm by 5 nm by 5 nm) AuNRs in sodium dodecyl sulfate was purchased from Nanopartz Inc. Long single-stranded scaffold DNA m13mp18 was purchased from Bayou Biolabs. UV cross-linkable ^CNVK phosphoramidite was purchased from Glen Research. The single-stranded DNA sticky ends containing ^CNVK were synthesized on an Applied Biosystems 394 DNA synthesizer. The remaining DNA strands were purchased from Integrated DNA Technology (IDT) Inc. The sticky end strands were purified using denaturing polyacrylamide gel electrophoresis. The staple strands with thiol group were purified by IDT using high-performance liquid chromatography.

Methods: DNA origami monomers were made by mixing scaffold strands m13mp18 with the staple strands at a molar ratio of 1:8 in 1× TAE/Mg²⁺ buffer. The mixture was placed in a thermocycling incubator through the following ramping procedure. The products were purified in 100-MW centrifugal filter to remove excess staple strands. DNA nanorobot was prepared by mixing robot-A, robot-B, and robot-C monomers at 53°C and cooled to 16°C at a rate of 0.7°C/hour. It was UV–cross-linked using the 365-nm UV light-emitting diode (LED) light (M365LP1^c, Thorlabs Inc.; power intensity, 24 mW/mm²) at 16°C for 20 min. The fabrication of chiroptical Au nanostructure was following Fig. 1F.

Results: Fabrication of 3D DNA industrial nanorobots: Our 3D fabrication process starts with the construction of the nanorobot, a cube corner–shaped DNA origami that is made by self-assembly, folding, and covalent bonding (Fig. 1A). The robot was made by hybridizing the complementary sticky ends on the origami monomer plates to form a 2D trimer and subsequently folding the semiflexible hinged plates 90° to fabricate a three-face cube corner using two pairs of strut strands—a short double-stranded DNA—on the adjacent planes (Fig. 1C). We further welded the plates to stabilize the 3D structure by the cycloaddition reaction between four pairs of photo–cross-linkable 3-cyanovinylcarazole (^CNVK), a pseudobase, through UV illumination (Fig. 1B). AFM images of the robots present a closed-looped trimer structure with the designed equal sign orientation, confirming the successful cross-linking and formation of the 3D robot (Fig. 1E). Production of chiral nanodevices using 3D DNA industrial nanorobots: Fig. 1F demonstrates the robot-controlled assembly of chiroptical trimer constructs from achiral AuNR-decorated origami plates. After grabbing the plates onto robot trimer, the programmed robot arms were designed to fold them 90° inward to form a cubic box. Thus, the local concentration of the plates was increased by ~10⁶, allowing their further hybridization and UV crosslinked. The three plates together could then form a trimer cube corner with the AuNRs surrounding the vertex in a right-handed manner. On heating, the product trimers could be released from the robot, and showed their difference in handedness in their circular dichroism, with inverted peaks at 720 and 800 nm (Fig. 1G).

Conclusion: Our novel DNA nanorobot represents a significant advancement in drug delivery technologies, offering a modular platform that can be enhanced with additional mechanical elements such as arms and folding struts for more intricate tasks. Here, we used only temperature as an actuator and ^CNVK and UV for welding. DNA hybridization/dehybridization can be controlled optically with different wavelengths by incorporation of photoisomers, like azobenzenes, and there are several ways of photo–cross-linking, such as cinnamate and psoralen. Therefore, 3D DNA industrial nanorobots can be made with many controllable degrees of freedom. With the capability to introduce multiple degrees of freedom, these 3D DNA industrial nanorobots can be customized with a variety of biocompatible materials—including lipids, peptides, polymers, and metals—making them versatile tools in nanomedicine. Specifically designed to be similar in size to organelles, these nanorobots can be efficiently internalized by cells to execute programmable tasks, providing a new avenue for targeted drug delivery that bypasses conventional pharmacokinetic barriers, thus enhancing therapeutic efficacy and specificity

References: Feng Zhou et al. ,Toward three-dimensional DNA industrial nanorobots.Sci. Robot.8,eadf1274(2023).DOI:10.1126/scirobotics.adf1274


Fabrication, processing, and chiral production using three dimensional industrial DNA nanorobot