Aptamer-Modified DNA Origami Nanopore for ATP Measurement
Authors: Akai Hiromu , Shoji Kan 
Published: December 2024

We report a DNA origami nanopore sensor that can detect a specific small biomolecule with a high S/N ratio. This sensor is constructed by trapping a single aptamer-modified DNA origami nanopore at the tip of a glass nanopipette, and target molecules are detected as the change in the ion currents caused by the interaction between target molecules and the aptamer modified at the entrance of the nanopore (Fig. 1). In this study, we prepared the aptamer-modified DNA origami nanopore having a suitable size and shape to be trapped at the tip of nanopipettes. Then, we demonstrated ATP detection using DNA-origami-nanopore-trapped nanopipettes and successfully obtained open/close signals whose open/close ratio was proportional to the ATP concentration. From these results, we believe that the DNA origami nanopore sensor will be a versatile method for the analysis of small biomolecules.
Hybrid nanopore systems that a DNA origami nanopore is trapped at the tip of a glass nanopipette have a potential to be applied as a novel DNA origami nanopore sensor because ion currents through DNA nanopores can be easily measured with high throughput.1 However, the detection of small molecules such as ATP is challenging because of the short translocation time of small molecules through the nanopore. Here, we proposed the DNA origami nanopore that can induce the large deformation in response to the small target molecule and developed the nanopore sensor that detect the deformation as the change in the ion currents. In detail, we designed the DNA origami nanopore that the ATP-binding aptamer is extended from the aperture of the nanopore. In this paper, we investigated the feasibility of ATP measurement with the hybrid nanopore system by trapping the DNA origami nanopore at the tip of the glass nanopipette and measuring the ion current through the nanopore.
To realize the concept, the nanopore that can be trapped at the tip of the nanopipette is required. Thus, we designed the DNA origami nanopore composed of a sheet and a barrel structure. By designing the larger sheet than the inner diameter of the nanopipette tip, the DNA origami nanopore can be physically trapped at the tip of the nanopipette. Moreover, the ATP-binding split DNA aptamer was extended from the edge of the barrel structure so that the aptamer-ATP complex can close the pore entrance (Fig. 2). First, we confirmed the formation and purification of the DNA nanopore by agarose gel electrophoresis. Before purification, two bands indicating the DNA nanopore and excess staple strands were observed. After purification, the excess staple strands were removed, and only the band of the DNA nanopore was observed (Fig. 3). From these results, we successfully formed and purified the aptamer-modified DNA origami nanopore.
Next, to investigate the possibility of trapping the DNA nanopore and measuring ATP, we performed ion current measurements of the hybrid nanopore system. First, we prepared electrolyte solutions containing DNA nanopores and ATP (0, 0.5, 1.0, and 2.0 mM) and inserted the nanopipette into the solution. Then, the positive potential was applied to trap the DNA nanopore and measure ion current through the trapped DNA origami nanopore (Fig. 4). Without ATP, stable ion current was observed following step-like current drop, indicating that the single DNA nanopore was trapped (Fig. 5a, b). Although the current drop around 1.8% was dominant, a high distribution was observed (Fig. 5c). This is because of the different positions of the trapped DNA nanopore. With ATP, the current signals that alternately repeat high and low currents were observed (Fig. 5a), and the all-point current histogram showed apparent two peaks (Fig. 5e), suggesting the binding and dissociating between the single aptamer and ATP. Moreover, analysis results of the open/close signals at each ATP concentration showed a linearly increased close probability (37.0 ± 28.2, 45.0 ± 10.5, and 68.3 ± 16.7%, Fig. 5f). Consequently, we confirmed that the DNA origami nanopore sensor can provide the open/close signals caused by the interaction between the aptamer and ATP with a high S/N ratio, and potentially measure ATP concentration. We believe that this technology can be applied to single-cell analysis, and provide unique insights into single-cell functions by integrating with the accurate manipulating technology of the glass nanopipette.
1. Hernández-Ainsa, S. and Keyser, U. F. nanoscale.
6, 14121–14132 (2014).

Figure 1