Albrecht Claire S, Scatena Lawrence F, von Hippel Peter H, Marcus Andrew H
Center for Optical, Molecular and Quantum Science, University of Oregon, Eugene, Oregon 97403.
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403.
Proc SPIE Int Soc Opt Eng. 2024 Jan-Feb;12863. doi: 10.1117/12.3001802. Epub 2024 Mar 13.
Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI) dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI) dimer-substituted DNA constructs.
碱基堆积对双链DNA的稳定性至关重要。然而，很少有实验能够直接探测DNA碱基的局部构象和构象波动。在此，我们报告一种新的光谱方法，使用可吸收紫外线的荧光鸟嘌呤类似物6-甲基异黄蝶呤（6-MI）来研究DNA碱基的局部构象，该类似物可用作位点特异性探针标记DNA。在这些实验中，我们将双光子激发（2PE）方法应用于二维荧光光谱（2DFS），这是一种荧光检测的非线性傅里叶变换光谱。在2DFS中，四个共线激光脉冲的重复序列（中心波长约675nm，相对相位以射频扫描）用于激发6-MI的最低能量电子 - 振动（振转）跃迁（中心波长约340nm）。随后产生的低通量荧光在单个光子水平上进行相位同步检测，并作为脉冲间延迟的函数。产生电子激发态布居的2PE跃迁途径包括电子基态和激发态之间的光学相干以及非共振（单光子激发）虚态。我们的结果表明，2PE-2DFS实验除了能提供有关（6-MI）二聚体内相邻6-MI单体之间构象相关的激子耦合信息外，还能提供有关6-MI单体电子 - 振动光谱的信息。原则上，这种方法可用于确定（6-MI）二聚体取代的DNA构建体的局部碱基堆积构象。
