Abstract: One-dimensional DNA nanomaterials exhibit broad potential for applications across multiple fields. However, quantitative evaluation of their self-assembly efficiency remains challenging. In this work, six-helix bundle (6HB) DNA fibers were employed as a model system. Building upon existing image analysis methods, we developed an integrated and parameterized approach to overcome the challenges of single-particle identification and counting in high-aspect-ratio, entangled one-dimensional fibers, and established an AFM image-based method for visualizing and quantifying self-assembly efficiency. In this method, the area coverage ratio and contour length density extracted from AFM images were used as quantitative parameters to characterize one-dimensional DNA fiber networks. Using this method, the effects of buffer pH (5–11) and Mg2? concentration (6.25–87.5 mM) on the assembly behavior of 6HB DNA fibers were investigated. The results show that efficient assembly of 6HB DNA fibers occurs within a well-defined window of conditions. Continuous long fibers are preferentially formed in the pH range of 6–8, with the highest assembly efficiency at pH 8, where the area coverage ratio reaches ~36%, and the contour length density is ~8 μm·μm?2. When the pH deviates from this range, the continuous one-dimensional network gradually transforms into fragmented or disordered aggregates, and both parameters decrease to background levels. Fiber assembly also shows a clear threshold response to Mg2? concentration: the assembly efficiency increases rapidly above 25 mM, and stable long fibers form at concentrations above 50 mM, corresponding to area coverage ratios >20% and contour length densities >4 μm·μm?2. These results quantitatively demonstrate the strong dependence of DNA nanostructure self-assembly on environmental conditions such as pH and ionic strength. The analytical framework established here provides a useful quantitative tool for the design, assembly, and application of one-dimensional DNA nanomaterials.

Key words: six-helix bundle DNA fibers, AFM image analysis, self-assembly efficiency