Cavendish Laboratory, University of Cambridge, Cambridge CB3 0HE, U.K.
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, United States.
ACS Nano. 2024 Jun 11;18(23):15013-15024. doi: 10.1021/acsnano.4c01466. Epub 2024 May 31.
Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.
电泳传输在推进传感技术方面起着关键作用。到目前为止,系统的研究集中在经典 B 型或 A 型核酸的转位上,而直接 RNA 分析正在成为纳米孔传感和测序的新前沿。在这里,我们将研究较少探索的非经典 RNA:DNA 杂交体在电泳传输中的动力学与研究充分的 B 型 DNA 传输进行比较。使用 DNA/RNA 纳米技术和固态纳米孔,研究了 RNA:DNA (RD) 和 DNA:DNA (DD) 双链体的易位。值得注意的是,尽管 RD 双链体包含相同数量的碱基对,但它们被发现比 DD 双链体更快地穿过纳米孔。我们的实验表明,RD 双链体呈现出一种非经典的螺旋结构,与 B 型 DD 分子具有不同的传输特性。我们发现,RD 和 DD 分子,具有相同的轮廓长度,以可比的速度通过纳米孔移动。我们使用原子力显微镜、原子分子动力学模拟、琼脂糖凝胶电泳和动态光散射测量来研究这两种双链体形式的物理特性。借助粗粒化和分子动力学模拟,我们发现电场对具有不同几何形状或形状的纳米孔中 RD 或 DD 双链体的片段施加的单位长度有效力大致相同。我们的结果阐明了螺旋形式在核酸转位中的重要性,对 RNA 传感、测序以及电泳传输的分子理解具有重要意义。