During the past decade, there has been a notable surge in novel analytical methods to assess single molecules, because of our increased interest in understanding potential similarities and differences in function and structure at the molecular level. Such techniques have provided researchers with valuable insights into dynamic processes such as temporal structural rearrangements and folding mechanisms. However, although researchers now routinely perform these techniques within research laboratories, their outcomes remain hit-and-miss since attaching a miniscule single molecule to a specific point on an addressable microscopic substrate is difficult to control, sometimes leading to two or more molecules occupying the same spot and resulting in less accurate analysis.

Reducing the concentration of molecules to try and overcome this problem lowers experimental throughput, thereby increasing time pressures, while increasing the concentration reduces data quality. High cost and complex manufacturing processes also play a part in the limitations of single-molecule research on bio-nanotechnology-chips. Increasing the effective size of molecules-of-interest by linking them to large DNA origami pegboards ensures that spatial manipulation can take place whilst maintaining an appropriate size to enable researchers to accurately position these constructs on surfaces without crowding or overlapping.

Moreover, by easily accessing the assembled molecular components on DNA origami pegboards, researchers can microscopically interrogate these molecules while retaining their functionality during studies. This allows scientists to visualise distinct sections of individual molecules to evaluate functionality and structure, hinting at the wider mechanisms at play. Leveraging self-assembly processes to create both the molecular constructs and their attachment surfaces from scratch using “bottom-up” techniques at the nanoscale increases the practicality of using large-scale single-molecule analysis methods since this can make dealing with extremely small components much more deterministic than it currently is.

To overcome the recognised complexities of currently available single-molecule experimental methods, corresponding authors, Drs. Rishabh Shetty, Rizal Hariadi, and Ashwin Gopinath at the Biodesign Institute at Arizona State University, CalTech, and MIT in the USA have recently designed a pioneering technique which facilitates the study of single molecules using precision positioning onto the selected surface without the need for expensive equipment and intensive training.

The researchers constructed a large grid of tiny two-dimensional structures (known as attachment areas) capable of containing the molecule of interest by using the highly desirable chemical and physical properties of self-assembling DNA origami pegboards. This meant that the team could place the constructs exactly where they wanted them on a pre-treated glass surface in a uniform manner to maximise the space used and increase the ease and accuracy of analysis.

In a critical aspect of this process, the researchers sought to identify the optimal binding site diameter required to attach the molecule to the surface. Each binding site was defined by creating a chemically attractive “attachment” surface for the DNA origami molecular construct. This process was highly parallelized by using the attractive and repulsive forces of self-assembly in nature that create hexagonally close-packed crystals from individual components (such as polystyrene nanospheres) on the meso-to-macroscale, which ranges from micrometres to millimetres.

They achieved the optimal binding site diameter by imaging the attachment footprint of polystyrene nanospheres of varying sizes using sophisticated microscopy techniques, discovering that the spacing between binding sites and the size of the binding sites themselves increased as the sphere size got bigger. Crucially, they also observed that each sphere within a hexagonal pattern touched its six nearest neighbours, a phenomenon inherent within the physical processes underlying the crystal structure self-assembly.

The authors used this to their advantage to define the size of each binding site (the effective footprint of the impinging polystyrene nanosphere) in addition to controlling the distance between adjacent sites for ease of microscopic analysis. Their observations highlighted the importance of matching the shape and size of the binding site with the DNA origami molecular construct to maintain the spatial arrangement and maximise single site occupancy. Following rigorous experiments, the researchers were able to pinpoint the ideal binding site size range to perfectly accommodate a single molecular construct of a specific size using their simplified method.

Once the researchers calculated appropriate binding site and molecule diameters, they coated tilted glass surfaces with a solution containing polystyrene nanospheres at a standardised temperature and humidity. On drying, a self-assembled closely packed hexagonal layer remained, which the researchers then treated with a chemical solution to create the attachment surfaces and rinsed in water to remove the polystyrene spheres. This created binding sites in a defined, grid-like spatial arrangement which were ready to receive the prefabricated single molecule DNA origami constructs.

The researchers then proceeded to carefully place the constructs onto the prepared hexagonal patterned surface to promote attachment, making sure that the optimal conditions which they had previously identified were implemented. They tweaked the system during the observation period to determine whether automated steps, alternative surface types, or different salt concentrations would alter adherence, to ensure that the system could be adapted if necessary and to more closely mimic physiological conditions.

Following the experimental incubation period, the team dehydrated the glass surfaces and then captured microscopic images for quality control and to determine their suitability for experimental analysis. They confirmed that their method produced images of comparable quality to those obtained using sophisticated conventional single molecule experimental techniques when rehydrated, even if high concentrations of the molecules of interest were present; something that could not be previously achieved in the field.

The research team demonstrated the efficiency and robustness of their method by comparing their results with those achieved using conventional techniques, showing 100% binding site occupation and double the amount of single-molecule binding than is possible in theory (the so-called Poisson limit of 37%) using conventional techniques. They also tested how long the molecules remained stably bound to the surface and discovered that it was possible to store the samples at room temperature for several months in the absence of any apparent degradation and without the need for a specialised storage environment. This assured the researchers that their method was viable for potential point-of-care applications on biochips.

Dr Shetty and colleagues have successfully developed and validated a robust, accessible, cost-effective method to enable researchers to study the structure and function of single molecules with substantially increased throughput and precision whilst maintaining high data quality. This work provides a platform for the accurate placement of molecular constructs onto several commercially available glass and semiconductor surfaces, which may be applied to numerous specialisms including biophysics, protein biochemistry, and digital molecular diagnostics. Furthermore, the high throughput process, prolonged shelf life, minimal training, and inexpensive equipment requirements drastically increase the potential scope and adoptability of this method.

This impressive feat offers a launchpad for the continued study of single molecules to increase our understanding of wider functional and structural molecular behaviours. Further optimisation of the preparation methods will undeniably increase the value of this technique for researchers and industry, potentially allowing people to use this model to manufacture large-scale variants at a hugely reduced cost.