Algar W Russ, Massey Melissa, Krull Ulrich J
Department of Chemical and Physical Sciences, University of Toronto at Mississauga, South Building, Room 2035, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada.
J Fluoresc. 2006 Jul;16(4):555-67. doi: 10.1007/s10895-006-0091-y. Epub 2006 Jun 23.
Fluorescence resonance energy transfer (FRET) was investigated between the intercalating dye thiazole orange (TO), and the dyes Cyanine 3 (Cy3), Cyanine 5 (Cy5), Carboxytetramethyl Rhodamine (TAMRA), Iowa Black FQ (IabFQ), and Iowa Black RQ (IabRQ), which were covalently immobilized at the end of dsDNA oligonucleotides. In addition to determining that TO was an effective energy donor, FRET efficiency data obtained from fluorescence lifetime measurements indicated that TO intercalated near the middle of the 19mer oligonucleotide sequence that was used in this study. Discrepancies in FRET efficiencies obtained from intensity and lifetime measurements led to the investigation of non-fluorescent complex formation between TAMRA and modified TO. The hydrophobicity of TO was modified by the addition of either an alkyl or polyethylene glycol (PEG) side-chain to study effects of dimer and aggregate formation. It was found that at stoichiometric excesses of modified TO, fluorescence quenching of TAMRA was observed, and that this could be correlated to the hydrophobicity of a TO-chain species. The TAMRA:TO-chain association constant for the TO-alkyl system was 0.043+/-0.002 M(-1), while that obtained for the TO-PEG was 0.037+/-0.002 M(-1). From the perspective of method development for the transduction of hybridization events, we present and evaluate a variety of schemes based on energy transfer between TO and an acceptor dye, and discuss the implications of complex formation in such schemes.
研究了嵌入染料噻唑橙（TO）与共价固定在双链DNA寡核苷酸末端的染料菁3（Cy3）、菁5（Cy5）、羧基四甲基罗丹明（TAMRA）、艾奥瓦黑FQ（IabFQ）和艾奥瓦黑RQ（IabRQ）之间的荧光共振能量转移（FRET）。除了确定TO是一种有效的能量供体外，通过荧光寿命测量获得的FRET效率数据表明，TO嵌入了本研究中使用的19聚体寡核苷酸序列的中间附近。从强度和寿命测量获得的FRET效率差异导致了对TAMRA与修饰后的TO之间非荧光复合物形成的研究。通过添加烷基或聚乙二醇（PEG）侧链来修饰TO的疏水性，以研究二聚体和聚集体形成的影响。结果发现，在修饰后的TO化学计量过量时，观察到TAMRA的荧光猝灭，并且这可能与TO链物种的疏水性相关。TO-烷基体系的TAMRA:TO链缔合常数为0.043±0.002 M⁻¹，而TO-PEG体系的缔合常数为0.037±0.002 M⁻¹。从杂交事件转导的方法开发角度出发，我们提出并评估了基于TO与受体染料之间能量转移的各种方案，并讨论了此类方案中复合物形成的影响。
