NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4.
NMR studies of triple-strand formation from the homopurine-homopyrimidine deoxyribonucleotides d(GA)4 and d(TC)4.
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高嘌呤-同型嘧啶脱氧核糖核苷酸 d(GA)4 和 d(TC)4 三链形成的 NMR 研究。
DOI：
10.1021/bi00445a048
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Feigon,J
中科院分区：
文献类型：
--
作者：
Rajagopal,P;Feigon,J
材料和方法样品制备。 DNA 寡核苷酸通过磷酸三酯法合成，并在 Jacques van Boom 博士的实验室中按照先前所述进行纯化（van der Marel 等人，1981），或者使用 ß-cynano-乙基亚磷酰胺和 µ 柱在 Applied Biosystems 381A DNA 合成仪上合成。后一种 DNA（用于大多数样品）按照 Kintanar 等人（1987）的方法通过凝胶过滤进行纯化。纯化的寡核苷酸在变性聚丙烯酰胺凝胶上以单条带形式运行。样品浓度通过 A260 测定，对 d (GA) 4 和 d (TC) 4 分别使用 9500 L mol'1 cm" 1 和 7500 L mol'1 cm" 1 的消光系数（Lee 等人，1979；Antao 等人，1988），并且根据样品每条链浓度为 2-6 mM。除非图例中另有说明，否则双链样品是通过混合等摩尔量的在适当的缓冲液中将两条链在 pH 7.3 中冻干，再溶解在 D20 中，再冷冻干燥，再溶解在 D20 中，然后转移到 NMR 管中，在 N2 (g) 流下干燥样品，然后将样品重新溶解在 400 µ 99.996% D20 中。通过添加溶解在 H2G 或 D20 中的适量第三链，然后通过向双链体样品中添加 HCl 将 pH 降低至 6.1 或 5.5，然后将这些样品重新干燥并重新溶解在其中含有 H8 的 400 µ D20 或 90% H2O/10% D20 中。质子被氘核取代的制备方法是通过将 d (GA) 4 链在 95°C 下加热 12 小时，导致 AH8 和 GH8 质子完全交换（Schweizer 等，1964），然后按上述方法制备三链体。
Materials and MethodsSample Preparation. DNA oligonucleotides were synthesized by the phosphotriester method and purified in the laboratory of Dr. Jacques van Boom as previously described (van der Marel et al., 1981), or they were synthesized on an Applied Biosystems 381A DNA synthesizer using ß-cynano-ethyl phosphoramidites and-µ columns. The latter DNA (used for most of the samples) was purified by gel filtration followingthe method of Kintanar et al.(1987).Purified oligonucleotides ran as single bands on denaturing polyacrylamide gels. Sample concentrations were determined by A260 by using extinction coefficients of 9500 and 7500 L mol'1 cm" 1 for d (GA) 4 and d (TC) 4, respectively (Lee et al., 1979; Antao et al., 1988) and were 2-6 mM in each strand depending on the sample. Unless otherwise indicated in the figure legend, duplex samples were prepared by mixing equimolar amounts of the two strands at pH 7.3 in the ap-propriate buffer. The sample was lyophilized, redissolved in D20, relyophilized, redissolved in D20, and transferred to a NMR tube. Samples were dried in the NMR tube under a stream of N2 (g) and redissolved in 400 µ of 99.996% D20. Spectra of the same samples in water were obtained after redrying thesamples and redissolving in 90% H2O/10% D20. Samples containing triplex were prepared by adding appropriate amounts of the third strand dissolved in H2G or D20 and then lowering the pH to 6.1 or 5.5 by addition of HC1 to the duplex samples. These samples were then redried and redissolved in 400 µ of D20 or 90% H2O/10% D20. Samples in which the H8 protonshad been replaced by deuterons were prepared by heating the d (GA) 4 strand at 95 C for 12 h, resulting in complete exchange of the AH8 and GH8 protons (Schweizer et al., 1964), and then the triplex was prepared as above.
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