High-speed 3D DNA-PAINT and unsupervised clustering for unlocking 3D DNA origami cryptography

Abstract DNA origami cryptography, which employs nanoscale steganography to conceal information within folded DNA origami nanostructures, shows promise as a secure molecular cryptography technique due to the large 700-bit key size generated through scaffold routing and sliding and the interlacing of staple strands. 1 However, achieving the promised security, high information density, fast pattern detection, and accurate information readout requires even more secure cryptography and fast readout. Here, we advance the DNA origami cryptography protocol by demonstrating its ability to encrypt specific information in both 2D and 3D DNA origami structures, thus increasing the number of possible scaffold routings and improving the encryption key size. To this end, we used all-DNA-based steganography, enabled by high-speed 2D and 3D DNA-PAINT super-resolution imaging, which does not require protein binding to reveal the pattern, allowing for higher information density. We combined 2D and 3D DNA-PAINT data with unsupervised clustering, achieving up to 89% accuracy and high ratios of correct-to-wrong readout despite significant flexibility in the 3D DNA origami structure shown by oxDNA simulation. Furthermore, we propose design criteria that ensure complete information retrieval for the DNA origami cryptography protocol. We anticipate that this technique will be highly secure and versatile, making it an ideal solution for secure data transmission and storage via DNA.