N. N. Semenov Institute of Chemical Physics RAS, Kosygina 4, 119991 Moscow, Russia.
Langmuir. 2010 Sep 21;26(18):14766-73. doi: 10.1021/la102472u.
To use the TRXF technique for the quantification of DNA binding to monolayers at the air-water interface, DNA from salmon testes was labeled by covalently bound bromine. For this purpose, an analytical procedure for the quantification of bromine in labeled DNA with a detection limit of 10-20 μg was developed. It was found that the pH of the solution has a strong influence on the yield of brominated DNA (BrDNA) when Br(2) is used as a reagent. Much higher degrees of bromination can be achieved at pH 5 than at pH 7. A degree of bromination above a threshold of 2 to 3% (bromine per base) leads to the cross linking of BrDNA with the formation of an insoluble gel during the precipitation procedure. Finally, a reaction scheme with N-bromosuccinimide (NBS) that avoids precipitation has been established. Succinimide and some bromide ions remain in the solution as byproducts. However, these bromide ions are not competitive with BrDNA for binding at positively charged monolayers. Therefore, a new method for binding studies of model DNA to Langmuir monolayers at the air-water interface has been established. An important result of these studies is the finding that higher salt concentrations (representing physiological conditions) lead to an increased amount of adsorbed DNA. This can be explained by the decrease in the effective charge of the DNA molecules with decreasing Debye screening length.
为了使用 TRXF 技术在气-水界面的单层上定量 DNA 与单层的结合，鲑鱼精巢的 DNA 被共价结合的溴原子标记。为此，开发了一种用于定量标记 DNA 中溴的分析方法，检测限为 10-20μg。研究发现，当使用 Br2 作为试剂时，溶液的 pH 值对溴化 DNA（BrDNA）的产率有很强的影响。在 pH 5 时，溴化程度比在 pH 7 时高得多。溴化程度超过 2%至 3%（每个碱基的溴）的阈值会导致 BrDNA 交联，在沉淀过程中形成不溶性凝胶。最后，建立了一种使用 N-溴代丁二酰亚胺（NBS）的避免沉淀的反应方案。副产物中有丁二酰亚胺和一些溴化物离子留在溶液中。然而，这些溴化物离子不会与带正电荷的单层上的 BrDNA 竞争结合。因此，建立了一种新的方法用于研究模型 DNA 与气-水界面 Langmuir 单层的结合。这些研究的一个重要结果是发现，较高的盐浓度（代表生理条件）会导致吸附的 DNA 量增加。这可以通过随着德拜屏蔽长度的减小 DNA 分子的有效电荷减小来解释。
