Perets Ethan A, Olesen Kristian B, Yan Elsa C Y
Department of Chemistry, Yale University, New Haven, Connecticut 06520, United States.
Langmuir. 2022 May 10;38(18):5765-5778. doi: 10.1021/acs.langmuir.2c00365. Epub 2022 Apr 28.
Many DNA-based technologies involve the immobilization of DNA and therefore require a fundamental understanding of the DNA structure-function relationship at interfaces. We present three immobilization methods compatible with chiral sum frequency generation (SFG) spectroscopy at interfaces. They are the "anchor" method for covalently attaching DNA on a glass surface, the "island" method for dropcasting DNA on solid substrates, and the "buoy" method using a hydrocarbon moiety for localizing DNA at the air-water interface. Although SFG was previously used to probe DNA, the chiral and achiral SFG responses of single-stranded and double-stranded DNA have not been compared systemically. Using the three immobilization methods, we obtain the achiral and chiral C-H stretching spectra. The results introduce four potential applications of chiral SFG. First, chiral SFG gives null response from single-stranded DNA but prominent signals from double-stranded DNA, providing a simple binary readout for label-free detection of DNA hybridization. Second, with heterodyne detection, chiral SFG gives an opposite-signed spectral response useful for distinguishing native (D-) right-handed double helix from non-native (L-) left-handed double helix. Third, chiral SFG captures the aromatic C-H stretching modes of nucleobases that emerge upon hybridization, revealing the power of chiral SFG to probe highly localized molecular structures within DNA. Finally, chiral SFG is sensitive to macroscopic chirality but not local chiral centers and thus can detect not only canonical antiparallel double helix but also other DNA secondary structures, such as a poly-adenine parallel double helix. Our work benchmarks the SFG responses of DNA immobilized by the three distinct methods, building a basis for new chiral SFG applications to solve fundamental and biotechnological problems.
许多基于DNA的技术都涉及DNA的固定化，因此需要从根本上理解界面处DNA的结构-功能关系。我们介绍了三种与界面处的手性和频产生（SFG）光谱兼容的固定化方法。它们分别是将DNA共价连接到玻璃表面的“锚定”方法、将DNA滴铸在固体基质上的“岛屿”方法以及使用烃基部分将DNA定位在空气-水界面的“浮标”方法。虽然SFG以前曾用于探测DNA，但尚未对单链和双链DNA的手性和非手性SFG响应进行系统比较。使用这三种固定化方法，我们获得了非手性和手性C-H伸缩光谱。结果介绍了手性SFG的四个潜在应用。第一，手性SFG对单链DNA给出零响应，但对双链DNA给出显著信号，为DNA杂交的无标记检测提供了一种简单的二元读数。第二，通过外差检测，手性SFG给出符号相反的光谱响应，可用于区分天然（D-）右手双螺旋和非天然（L-）左手双螺旋。第三，手性SFG捕获杂交时出现的核碱基的芳香族C-H伸缩模式，揭示了手性SFG探测DNA内高度局部化分子结构的能力。最后，手性SFG对宏观手性敏感，但对局部手性中心不敏感，因此不仅可以检测标准的反平行双螺旋，还可以检测其他DNA二级结构，如聚腺嘌呤平行双螺旋。我们的工作为通过三种不同方法固定化的DNA的SFG响应建立了基准，为解决基本问题和生物技术问题的新手性SFG应用奠定了基础。
