Autonomous Molecular Crawlers for Spatial Analysis via DNA Nanotechnology

By Soham NandiReviewed by Susha Cheriyedath, M.Sc.Apr 26 2024

In an article published in the journal Nature, researchers introduced a molecular robotic system utilizing deoxyribonucleic acid (DNA)-based artificial motors, termed 'crawlers', to autonomously survey molecular environments and report spatial information. These crawlers copied information from DNA-labeled targets, generating records reflecting their trajectories.

The system demonstrated the ability to count subunits in molecular complexes and detect multivalent proximities, aiding in the creation of detailed molecular interaction maps inside cells with nanoscale resolution.

Background

The field of synthetic molecular motors has advanced considerably, offering insights into natural motor functions and potential applications in artificial life forms. DNA, with its programmability and compatibility with biological systems, serves as an ideal material for designing molecular motors. Previous developments in DNA-based motors have ranged from simple walkers to complex robots capable of performing various tasks. However, existing methods have limitations in surveying molecular environments autonomously and repeatedly, hindering comprehensive spatial analysis at the molecular scale.

This paper addressed these gaps by introducing a molecular robotic system composed of 'crawlers' that roam molecular landscapes, collecting spatial information from DNA-labeled targets. Unlike previous methods, these crawlers operated autonomously and could count subunits in molecular complexes while detecting multivalent proximity interactions.

By employing a mechanism that allowed random crawling and information copying from targets, this system offered non-destructive, repeated sampling of molecular environments. This innovation bridged the gap in current methodologies, enabling detailed examination of spatial arrangements at the molecular scale, essential for refining studies of spatial transcriptomes and proteomes. Through the development of this molecular robotic system, the paper aimed to facilitate the creation of large-scale molecular interaction maps with nanoscale resolution inside cells.

Employing DNA Nanotechnology for Molecular Recording and Cellular Imaging

DNA origami, a promising nanotechnology, was employed to construct intricate structures using DNA strands. Oligonucleotides were meticulously designed and obtained from Integrated DNA Technologies (IDT). The origami scaffold strand, pivotal for structure assembly, was procured from Bayou Biolabs.

To prepare the origami, a meticulous annealing process was conducted, involving controlled temperature reduction. Streptavidin samples were prepared by incubating probe duplexes with streptavidin at specific concentrations. Cell samples were meticulously treated for colocalization tests, involving multiple steps including fixation, permeabilization, and blocking. Antibody-DNA conjugation was achieved through a precise chemical reaction between thiol-modified DNA oligonucleotides and antibodies.

Crawler recording reactions on DNA origami were conducted using Bst polymerase and specific primer mixes. Polymerase chain reaction (PCR) amplification and gel electrophoresis were employed to analyze the recorded data. Sanger sequencing and high-throughput sequencing provided further insights into the recorded information. Fluorescence microscopy was utilized for imaging cell samples after recording reactions. Statistical analysis was conducted to evaluate the significance of experimental results.

Various software tools were employed for data acquisition, analysis, and visualization, ensuring comprehensive investigation and interpretation of findings. Overall, the researchers employed a meticulous approach to explore the potential of DNA nanotechnology in diverse applications, ranging from molecular recording to cellular imaging.

Key Findings and Demonstrations

The authors presented a novel approach utilizing DNA nanotechnology to design a molecular crawler system for molecular recording and cellular imaging. The crawler system consisted of probe molecules with primer-binding and copy-and-release domains, enabling autonomous growth and release along designated tracks. Demonstrations on DNA origami platforms confirmed the scalability and functionality of the crawler system, showcasing its ability to accurately record and retrieve molecular information repeatedly.

Furthermore, the system exhibited random crawling behavior, allowing for the detection and counting of multivalent interactions within molecular complexes. This capability was demonstrated through experiments involving streptavidin complexes and artificial molecular structures, illustrating the system's versatility and potential for high-throughput applications. In a biological context, the crawler system was applied to detect trivalent protein interactions at microtubule growing ends within fixed cells.

Perturbation experiments revealed the system's ability to distinguish different cellular states based on protein colocalization patterns, with gel analysis and fluorescence microscopy confirming the accuracy and reliability of the recorded data. Overall, the authors highlighted the molecular crawler system's robustness, scalability, and applicability in both controlled laboratory settings and complex biological environments. By offering a non-destructive and catalytic approach to molecular recording and detection, this technology opened up new avenues for exploring molecular interactions and cellular dynamics with high precision and efficiency.

Discussion on Molecular Robotic Agents

The discussion highlighted the potential of molecular robotic "agents" for quantitative analysis of molecular landscapes, showcasing their ability to amplify signals from low-concentration samples while keeping molecules intact. These agents, though individually simple, collectively provided insights beyond what a single agent could offer, such as determining the number of subunits in molecular complexes and assessing multivalent protein colocalization.

Future developments could enable swarm behaviors, where agents interact based on recorded instructions. The system's versatility in DNA tagging opened avenues for broader applications, including high-throughput analysis of low-copy-number proteins and potential studies of protein-DNA interactions and chromosome organization. Ultimately, with unique labeling and sequencing techniques, the system held promise for mapping molecular interactions within cells with nanoscale resolution, advancing quantitative biological studies significantly.

Conclusion

In conclusion, the introduction of molecular robotic "crawlers" marked a significant advancement in the field of molecular nanotechnology. With their ability to autonomously survey molecular landscapes, count subunits in complexes, and detect multivalent interactions, these crawlers offered a powerful tool for spatial analysis at the nanoscale.

Leveraging DNA nanotechnology, they enabled non-destructive sampling and provided insights crucial for understanding spatial transcriptomes and proteomes. Looking ahead, further developments in swarm behaviors and broader applications hold promise for unraveling intricate molecular interactions with unprecedented resolution, paving the way for transformative advancements in quantitative biological studies.