Gulyak Evgeny L, Brylev Vladimir A, Zhitlov Mikhail Y, Komarova Olga A, Ustinov Alexey V, Sapozhnikova Ksenia A, Alferova Vera A, Korshun Vladimir A, Gvozdev Daniil A
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, 119991 Moscow, Russia.
Molecules. 2024 Dec 27;30(1):57. doi: 10.3390/molecules30010057.
Sulfonated indocyanines 3 and 5 (sCy3, sCy5) are widely used to label biomolecules. Their high molar absorption coefficients and lack of spectral overlap with biopolymers make them ideal as linker components for rapid assessment of bioconjugate stoichiometry. We recently found that the determination of the sCy3:sCy5 molar ratio in a conjugate from its optical absorption spectrum is not straightforward, as the sCy3:sCy5 absorbance ratio at the maxima tends to be larger than expected. In this work, we have investigated this phenomenon in detail by studying the spectral properties of a series of sCy3-sCy5 conjugates in which the dyes are separated by linkers of various lengths, including DNA duplexes. It was found that when sCy3 and sCy5 are located in close proximity, they consistently exhibit an "abnormal" absorbance ratio. However, when the two dyes are separated by long rigid DNA-based spacers, the absorbance ratio becomes consistent with their individual molar absorption coefficients. This phenomenon should be taken into account when assessing the molar ratio of the dyes by UV-Vis spectroscopy.
磺化吲哚菁3和5（sCy3、sCy5）被广泛用于标记生物分子。它们高的摩尔吸收系数以及与生物聚合物缺乏光谱重叠，使其成为用于快速评估生物共轭物化学计量的理想连接子成分。我们最近发现，从共轭物的光吸收光谱确定其中sCy3:sCy5的摩尔比并非易事，因为在最大值处sCy3:sCy5的吸光度比往往比预期的要大。在这项工作中，我们通过研究一系列sCy3-sCy5共轭物的光谱特性详细研究了这一现象，其中染料通过包括DNA双链体在内的各种长度的连接子隔开。发现当sCy3和sCy5靠得很近时，它们始终表现出“异常”的吸光度比。然而，当两种染料被长的刚性DNA基间隔物隔开时，吸光度比变得与其各自的摩尔吸收系数一致。在通过紫外-可见光谱评估染料的摩尔比时应考虑这一现象。
