Ishkitiev Nikolay, Miteva Marina, Micheva Maria, Stoyanova Tanya, Lozanova Vesela V, Lozanov Valentin S, Mihaylova Zornitsa, Cheshmedzhieva Diana V, Kandinska Meglena, Rangelov Miroslav, Todorova Nadezhda, Ilieva Sonia, Baluschev Stanislav, Gargallo Raimundo, Calenic Bogdan, Constantinescu Ileana, Landfester Katharina, Vasilev Aleksey A
Medical University Sofia, Dept. of Medical Chemistry and Biochemistry, 2 Zdrave str., Sofia 1431, Bulgaria.
Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.
Int J Biol Macromol. 2023 Oct 1;250:126094. doi: 10.1016/j.ijbiomac.2023.126094. Epub 2023 Aug 5.
In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.
鉴于近期逆转录病毒大流行，发现用于研究和诊断的新型多样RNA特异性荧光染料的问题变得极为重要。绝大多数核酸特异性探针要么不染色RNA，要么无法区分DNA和RNA。聚甲炔染料的多功能性使其适合用作核酸、蛋白质和其他生物分子可视化、分析和检测的染色剂。我们合成了不对称双阳离子同二聚体单甲炔花青染料1,1'-(1,3-亚苯基双(亚甲基))双(4-((3-甲基苯并[d]噻唑-2(3H)-亚基)甲基)吡啶-1-鎓)溴化物（Т1）和1,1'-(1,3-亚苯基双(亚甲基))双(4-((3-甲基苯并[d]噻唑-2(3H)-亚基)甲基)喹啉-1-鎓)溴化物（M1），并测试了它们的结合特异性、光谱特性、在活细胞和固定细胞中的膜穿透性、细胞毒性以及荧光发射稳定性。间充质细胞具有多样的表型以及广泛的增殖和分化特性。我们发现染料Т1和M1在来自根尖乳头的活间充质干细胞（SCAP）中表现出高光化学稳定性，与核酸结合时具有强荧光信号。我们发现M1在体内DNA可视化方面比对照荧光染料（Hoechst 33342）表现更好。另一方面，T1在活细胞中染色类似于核糖体的颗粒状细胞结构，在核膜通透后染色核仁而非细胞核中的染色质。这使得T1适用于活细胞和固定细胞中富含RNA的结构的可视化。
