Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark.
J Phys Chem B. 2010 Jan 21;114(2):1050-6. doi: 10.1021/jp909471b.
The fluorescent nucleobase analogues of the tricyclic cytosine (tC) family, tC and tC(O), possess high fluorescence quantum yields and single fluorescence lifetimes, even after incorporation into double-stranded DNA, which make these base analogues particularly useful as fluorescence resonance energy transfer (FRET) probes. Recently, we reported the first all-nucleobase FRET pair consisting of tC(O) as the donor and the novel tC(nitro) as the acceptor. The rigid and well-defined position of this FRET pair inside the DNA double helix, and consequently excellent control of the orientation factor in the FRET efficiency, are very promising features for future studies of nucleic acid structures. Here, we provide the necessary spectroscopic and photophysical characterization of tC(nitro) needed in order to utilize this probe as a FRET acceptor in nucleic acids. The lowest energy absorption band from 375 to 525 nm is shown to be the result of a single in-plane polarized electronic transition oriented approximately 27 degrees from the molecular long axis. This band overlaps the emission bands of both tC and tC(O), and the Forster characteristics of these donor-acceptor pairs are calculated for double-stranded DNA scenarios. In addition, the UV-vis absorption of tC(nitro) is monitored in a broad pH range and the neutral form is found to be totally predominant under physiological conditions with a pK(a) of 11.1. The structure and electronic spectrum of tC(nitro) is further characterized by density functional theory calculations.
三环胞嘧啶（tC）家族的荧光核苷类似物，如 tC 和 tC（O），具有高荧光量子产率和单一荧光寿命，即使掺入双链 DNA 后也是如此，这使得这些碱基类似物特别适合作为荧光共振能量转移（FRET）探针。最近，我们报道了第一个由 tC（O）作为供体和新型 tC（硝化物）作为受体组成的全核苷 FRET 对。在 DNA 双螺旋内，这个 FRET 对的刚性和明确位置，以及在 FRET 效率中对取向因子的出色控制，是未来研究核酸结构的非常有前途的特征。在这里，我们提供了在核酸中用作 FRET 受体所需的 tC（硝化物）的必要光谱和光物理特性。从 375 到 525nm 的最低能量吸收带是由一个大约 27 度偏离分子长轴的平面偏振电子跃迁引起的。该带与 tC 和 tC（O）的发射带重叠，并且计算了这些供体-受体对在双链 DNA 情况下的福斯特特性。此外，在广泛的 pH 范围内监测 tC（硝化物）的紫外-可见吸收，并且发现中性形式在生理条件下完全占主导地位，pK（a）为 11.1。通过密度泛函理论计算进一步表征了 tC（硝化物）的结构和电子光谱。
