Lemos Tony, Kim Harold D
School of Physics, Georgia Institute of Technology, Atlanta, GA 30332-0430, USA.
ArXiv. 2025 Aug 20:arXiv:2508.15091v1.
A short (<150 bp) double-stranded DNA (dsDNA) molecule ligated end-to-end forms a DNA minicircle. Due to sequence-dependent, nonuniform bending energetics, such a minicircle is predicted to adopt a certain inside-out orientation, known as the poloidal orientation. Despite theoretical and computational predictions, experimental evidence for this phenomenon has been lacking. In this study, we introduce a single-molecule approach to visualize the poloidal orientation of DNA minicircles. We constructed a set of DNA minicircles, each containing a single biotin located at a different position along one helical turn of the dsDNA, and imaged the location of biotin-bound NeutrAvidin relative to the DNA minicircle using atomic force microscopy (AFM). We applied this approach to two DNA sequences previously predicted to exhibit strongly preferred poloidal orientations. The observed relative positions of NeutrAvidin shifted between the inside and outside of the minicircle with different phases, indicating distinct poloidal orientations for the two sequences. Coarse-grained simulations revealed narrowly distributed poloidal orientations with different mean orientations for each sequence, consistent with the AFM results. Together, our findings provide experimental confirmation of preferred poloidal orientations in DNA minicircles, offering insights into the intrinsic dynamics of circular DNA.
一个短的（<150碱基对）双链DNA（dsDNA）分子首尾相连形成一个DNA微环。由于序列依赖性的、不均匀的弯曲能量学，预计这样的微环会采取一种特定的由内向外的取向，即极向取向。尽管有理论和计算预测，但一直缺乏这种现象的实验证据。在本研究中，我们引入了一种单分子方法来可视化DNA微环的极向取向。我们构建了一组DNA微环，每个微环在dsDNA的一圈螺旋上的不同位置含有一个生物素，并使用原子力显微镜（AFM）对生物素结合的中性抗生物素蛋白相对于DNA微环的位置进行成像。我们将这种方法应用于先前预测会表现出强烈偏好极向取向的两个DNA序列。观察到的中性抗生物素蛋白的相对位置在微环的内部和外部之间以不同相位移动，表明这两个序列有不同的极向取向。粗粒度模拟显示每个序列的极向取向分布狭窄且平均取向不同，这与AFM结果一致。总之，我们的发现为DNA微环中偏好的极向取向提供了实验证实，为环状DNA的内在动力学提供了见解。
