Zamotaiev Oleksandr M, Postupalenko Viktoriia Y, Shvadchak Volodymyr V, Pivovarenko Vasyl G, Klymchenko Andrey S, Mély Yves
Department of Chemistry, Kyiv National Taras Shevchenko University, 01033 Kyiv, Ukraine.
Bioconjug Chem. 2011 Jan 19;22(1):101-7. doi: 10.1021/bc100434d. Epub 2010 Dec 21.
Environmentally sensitive labels constitute a new, attractive tool for monitoring biomolecular interactions. 3-Hydroxychromone derivatives are of particular interest because they undergo excited-state intramolecular proton transfer (ESIPT) showing dual emission highly sensitive to environmental hydration. To overcome the drawbacks of the previously developed label for sensing protein-DNA interactions based on 2-furanyl-3-hydroxychromone (FC), a series of hydration-sensitive labels based on 3-hydroxy-4'-methoxyflavone have been synthesized. As compared to FC, the new labels display higher sensitivity of the ratio of their two emission bands (N*/T*) to solvent polarity and H-bond donor ability, as well as higher fluorescence quantum yields in water. Moreover, they show higher pK(a) values of their 3-hydroxyl group, allowing their application at neutral pH without interference of anionic forms. To illustrate the applications of these labels, we covalently coupled them to the N-terminus of the Tat(44-61) peptide that corresponds to the basic domain of the HIV-1 Tat protein. This coupling did not modify the nucleic acid chaperone properties of the peptide. Binding of oligonucleotides of varying length, sequence, and strandedness to the labeled peptides induced dramatic change in the N*/T* ratio of their two emission bands. This change indicated that the level of probe hydration in the peptide/oligonucleotide complexes decreases in the following order: short ssDNAs ≫ long ssDNAs > DNA hairpins > dsDNAs. The level of probe hydration was related to the ability of the probe to stack with the DNA bases or base pairs in the various complexes. The changes in the N*/T* ratio upon interaction of the labeled Tat peptides with DNA were about 3-fold larger with the new probes as compared to the parent FC label, in line with the higher sensitivity of the new probes to the environment. One of these labels, presenting the most compact geometry, showed the highest sensitivity, probably due to its optimal stacking with the DNA bases. Thus, the new hydration-sensitive labels appear as improved highly sensitive tools to site-selectively monitor the binding of peptides to oligonucleotides and nucleic acids.
对环境敏感的标签构成了一种用于监测生物分子相互作用的新型、有吸引力的工具。3-羟基色酮衍生物特别受关注，因为它们会发生激发态分子内质子转移（ESIPT），表现出对环境水合作用高度敏感的双重发射。为了克服先前开发的基于2-呋喃基-3-羟基色酮（FC）的用于检测蛋白质-DNA相互作用的标签的缺点，已经合成了一系列基于3-羟基-4'-甲氧基黄酮的水合敏感标签。与FC相比，新标签的两个发射带的比率（N*/T*）对溶剂极性和氢键供体能力表现出更高的灵敏度，并且在水中具有更高的荧光量子产率。此外，它们的3-羟基基团显示出更高的pK(a)值，使其能够在中性pH下应用而不受阴离子形式的干扰。为了说明这些标签的应用，我们将它们共价偶联到与HIV-1 Tat蛋白的碱性结构域相对应的Tat(44-61)肽的N末端。这种偶联没有改变该肽的核酸伴侣性质。不同长度、序列和链状结构的寡核苷酸与标记肽的结合导致其两个发射带的N*/T比率发生显著变化。这种变化表明，肽/寡核苷酸复合物中探针的水合水平按以下顺序降低：短单链DNA≫长单链DNA>DNA发夹结构>双链DNA。探针的水合水平与探针在各种复合物中与DNA碱基或碱基对堆积的能力有关。与母体FC标签相比，标记的Tat肽与DNA相互作用时N/T*比率的变化用新探针大约大3倍，这与新探针对环境的更高灵敏度一致。这些标签中的一个呈现出最紧凑的几何形状，显示出最高的灵敏度，这可能是由于它与DNA碱基的最佳堆积。因此，新的水合敏感标签似乎是用于位点选择性监测肽与寡核苷酸和核酸结合的改进的高灵敏度工具。
