Solution structure of poly(dA-dT).poly(dA-dT) in low and high salt: a 500 MHz 1H NMR study using one-dimensional NOE.
Solution structure of poly(dA-dT).poly(dA-dT) in low and high salt: a 500 MHz 1H NMR study using one-dimensional NOE.
复制标题
低盐和高盐中聚 (dA-dT).聚(dA-dT) 的溶液结构：使用一维 NOE 的 500 MHz 1H NMR 研究。
DOI：
10.1080/07391102.1984.10507529
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发表时间：
1984
影响因子：
4.4
通讯作者：
Sarma,RH
中科院分区：
文献类型：
--
作者：
Sarma,MH;Gupta,G;Sarma,RH
聚（dA-dT）·聚（dA-dT）在低盐（10-100 mM NaCl）和高盐（4-6 M CsF）中的CD光谱是不同的，即275 nm谱带在从低盐到高盐的过程中发生反转（Vorhickova et. al.MarJ.摩尔166，85，1983）。然而，仅从CD光谱不可能破译聚（dA-dT）·聚（dA-dT）的低盐和高盐形式之间可能存在的任何结构差异。因此，我们求助于高分辨率NMR光谱来了解poly（dA-dT）· poly（dA-dT）在低盐和高盐下的结构性质。通过对屏蔽常数的详细分析和在最小自旋扩散条件下使用C（8）-氘代poly（dA-dT）· poly（dA-dT）的NOE研究的广泛使用，我们得出了以下结论：（i）在低盐和高盐条件下，碱基配对是Watson-Crick，（ii）在盐的两种条件下，实验数据可以用左右手B-DNA双链体与左-惯用手占70%，惯用右手占30%。在400个碱基对长的poly（dA-dT）· polyidA-dT）（如本研究中所用），右手螺旋和左手螺旋之间的平衡也可以意味着在同一分子中存在两个螺旋结构域，这些结构域之间快速交换或/和这些结构域沿着螺旋轴不受阻碍的运动/传播。（iii）然而，聚（dA-dT）·聚（dA-dT）的低盐和高盐形式之间存在其他结构差异;在低盐条件下，右旋和左旋B-DNA双链体具有单核苷酸肽作为结构重复，而在高盐条件下，右旋和左旋B-DNA双链体具有二核苷酸作为结构重复。（iv）盐（CsF）诱导的poly（dA-dT）· poly（dA-dT）的结构转变没有任何沃森-克里克配对的破坏，（v）poly（dA-dT）· poly（dA-dT）的高盐形式不是左手Z-螺旋。仍然存在一个问题，即盐（CsF）诱导的poly（dA-dT）· poly（dA-dT）的CD光谱的变化真正表明了什么？有趣的是，我们可以表明，盐（CsF）诱导的poly（dA-dT）· poly（dA-dT）的变化与由碱性多肽即poly-L（Lys 2-Ala）引起的变化非常相似。这两种试剂都诱导DNA中的β-结构。为了减少自旋扩散引起的并发症，我们采用了很小的预饱和脉冲长度和400 ± 150 bp的C（8）-氘代poly（dA-dT）· poly（dA-dT）。尽管扩散的主要位点如C（8）H的氘化显著降低了扩散，但为了确保我们的结论不会受到在短预饱和时间下如此长的片段中可能的扩散的影响，我们使用d（A-T-A-T-A-T）的六个碱基对长双链体重复了我们的实验，发现结果与聚合物的结果惊人地相似。
CD spectra of poly(dA-dT)· poly(dA-dT) in low salt (10–100 mM NaCl) and high salt (4–6 M CsF) are different i.e. 275 nm band gets inverted in going from low to high salt (Vorhickova et. al.MarJ. Mol. Biol. 166, 85, 1983). However, from CD spectra alone it is not possible to decipher any structural differences that might exist between the low and high salt forms of poly(dA-dT)• poly(dA-dT). Hence, we took recourse to high resolution NMR spectroscopy to understand the structural properties of poly(dA-dT)• poly(dA-dT) in low and high salt. A detailed analysis of shielding constants and extensive use of NOE studies under minimum spin diffusion conditions using C(8)-deuterated poly(dA-dT)• poly(dA-dT) enabled us to come up with the following conclusions (i) base-pairing is Watson-Crick under low and high salt conditions, (ii) under both the conditions of salt the experimental data can be explained in terms of an equilibrium blend of right and left-handed B-DNA duplexes with the left-handed form 70% and the right-handed 30%. In a 400 base pairs long poly(dA-dT)• polyidA-dT) (as used in this study), equilibrium between right and left-handed helices can also mean the existence of both helical domains in the same molecule with fast interchange between these domains or/and unhindered motion/propagation of these domains along the helix axis, (iii) However, there are other structural differences between the low and high salt forms of poly(dA-dT) • poly(dA-dT); under the low salt condition, right-and left-handed B-DNA duplexes have mononucleotide as a structural repeat while under the high salt conditions, right-and left-handed B-DNA duplexes have dinucleotide as a structural repeat. In the text we provide the listing of torsion angles for the low and high salt structural forms, (iv) Salt (CsF) induced structural transition in poly(dA-dT)• poly(dA-dT) occurs without any breakage of Watson- Crick pairing, (v) The high salt form of poly(dA-dT)• poly(dA-dT) is not the left-handed Z-helix.Although the results above from NMR data are quite unambiguous, a question still remains i.e. what does the salt (CsF) induced change in the CD spectra of poly(dA-dT)• poly(dA-dT) really indicate? Interestingly, we could show that the salt (CsF) induced change in poly(dA-dT)• poly(dA-dT) is quite similar to that caused by a basic polypeptide viz. poly-L(Lys2-Ala)ni.e. both the agents induced a ψ-structure in DNA. And it was also demonstrated that the changes in poly(dA-dT)• poly(dA-dT) as caused by CsF and poly-L-(Lys2-Ala)ncould be reverted back by ethidium bromide-a relaxing agent.To minimize complications from spin diffusion in this study we have used very small presaturation pulse lengths and C(8)-deuterated poly(dA-dT)• poly(dA-dT) of 400 ± 150 bp long. Even though deuteration of a primary site of diffusion such as C(8)H substantially decreases diffusion, in order to make sure that our conclusions are not compromised by possible diffusion in such a long fragment under small presaturation times, we have repeated our experiments using the six base pair long duplex of d(A-T-A-T-A-T) and found the results to be strikingly similar to that from the polymer.