DNA BENDING 31P DECOUPLED ECOSY: EXOCYCLIC TORSION IN DNA OLIGONUCLEOTIDE DUPLEX

DNA 弯曲 31P 解耦 ECOSY：DNA 寡核苷酸双链体中的外环扭转

• 批准号: 6118697
• 负责人: SANTHARAM KAMATH
• 金额: $ 2.78万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-15 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6118697
• 关键词: biomedical equipment development; biomedical resource; technology /technique

The conformation about the exocyclic C4'-C5'bond in the DNA backbone plays a crucial rule in the bending of DNA. The conformers about this bond can be represented as a rapid equilibrium mixture of the three classical rotamers, gauche' (g' ), gauche- (g- ) and trans (t). For example, transition from the gauche+ conformer to the trans will increase the interphosphate distance between residues. This will cause local unstacking, thereby making the base pair roll into the major groove, creating a kink or hinge. In crystals, the overwhelmingly preferred conformation is g+ with a few instances of t, and g- is seldom observed. Examination of the P-P distances vis-a vis the C5'-C4' torsion in seventeen B-DNA, thirty two A-DNA and eleven RNA crystal structures with resolutions better than 2.5A reveal the following: 652 occurrences of g', and 54 of trans conformations about the C5'-C4' bond. The P-P distance in the trans conformer was always found to be larger than what was in the g'conformer. However, attempts to determine the sequence dependence of DNA bending, by high resolution crystallography of DNA duplexes in the absence of proteins, are often thwarted by artifacts associated with crystal packing. The ECOSY technique was developed to directly extract small passive couplings in the presence of large active couplings. Because H5'/H5" is a geminal system, it should be possible to employ the ECOSY technique to extract the values for 'JH'-H5. and'JHI'-H5". However, there could be three problems associated with this approach. First, many of the ECOSY cross peaks between 114' and H5'/H5" are expected to remain very close to the diagonal. Second, the chemical shifts of H2' and H2" of the deoxyribose ring are in general significantly different from each other and situated far away from the corresponding Hl'; the magnitudes of JHI'-H2' and JHI'-H2" are large, in the range of 6 to 9 Hz. On the other hand, the H4'-H5'/H5" is a tightly coupled AB system with small couplings between H4'and H5'/H5". Thus, the ECOSY spectra will have to simulated in order'JH'.H5' and 3 JH4'-H5". Third, the chemical shift anisotropy of "P may cause considerable line broadening, making it difficult to extract the se small coupling constants. With respect to the first problem, higher frequencies may provide a partial solution; however this will aggravate the third problem. With respect to the second problem, we have examined computer generated ECOSY spectra for typical values for H F, H2'& H2" as well as for H4', H5' & H5". It is seen that for H2'/H2", there are seven lobes of equal intensity in which the cc and P spin states are well-displaced in co2 and just overlap in (o 1. For H5'/H5 " there are eight lobes, and the displacements of the spin states are opposite to what is obsereved for H2'/H2". This difference in appearance has to do with the magnitude of the geminal coupling vis-a-vis that of J12'512" and J4'5'/4'5". In the H5'/H5" system, the intensities of the outer 8 lobes are significantly less than the inner 8 lobes because it is a typical AB system. To investigate the effect of chemical shift anisotropy we recorded ECOSY spectra of the 11 mer duplex AAA at 500, 600 and 750 MHz NMR systems at different temperatures in the presence and absence of "P decoupling. In the absence of "P decoupling, the proton line width increases with increasing magnetic field strength. "P decoupling, on the other hand, leads to a spectacular improvement in the ECOSY spectra, which contain multiplets with 8 lobes displaying a typical AB pattern. Thus, we have shown that "P decoupled ECOSY spectra can be employed to unambigously extract the JH4'-H5' and JH4'-H5" coupling constants in DNA oligonucleotides. However, the method will be useful only for those residues that display their H4'-H5'/H5" cross peaks off the diagonal. Unfortunately, these conclusions were based on "P decoupled ECOSY experiments at 600 MHz. Our attempts to perform "P decoupled ECOSY at 750 Mhz resulted in the loss of signals from 4',5' and 5" region.

DNA环外C4 ′-C5 ′键的构象 骨架在DNA的弯曲中起着至关重要的作用。 构象异构体 关于这个键可以表示为一个快速平衡混合物， 三种经典的旋转异构体，gauche'（g'），gauche-（g-）和trans-gauche-（g-）， （t）. 例如，从gauche+构象转变为transform 将增加残基之间的磷酸盐间距离。 这将 导致局部解堆积，从而使碱基对滚入 主要凹槽，形成扭结或铰链。 在晶体中， 压倒性地优选的构象是G+， 而g-很少被观察到。 P-P距离的检验 17个B-DNA、32个A-DNA和11个C5 ′-C4 ′扭转 分辨率优于2.5A的RNA晶体结构揭示了 以下：652次g '，54次反式构象， C5-C4键。 反式构象中的P-P距离总是 发现比g '构象异构体中的大。 然而，在这方面， 试图确定DNA弯曲的序列依赖性，通过高 在没有蛋白质的情况下DNA双链体的分辨率晶体学， 常常被与晶体堆积有关的人工产物所阻碍。 的 ECOSY技术被开发用于直接提取小被动 在存在大的有源耦合的情况下的耦合。 因为H5 '/H5” 是一个双子系统，应该可以使用ECOSY 技术来提取'JH'-H5的值。 和“JHI”-H5”。 然而，在这方面， 这种方法可能有三个问题。 第一、 预计114 ′和H5 ′/H5 ″之间的许多ECOSY交叉峰 保持非常接近对角线。 第二，H2'的化学位移 脱氧核糖环的H2“和H2”通常显著不同， 彼此远离并且远离对应的H1 '; JHI '-H2'和JHI '-H2”的大小较大，在6到9之间 Hz.另一方面，H4 '-H5'/H5”是一个紧耦合的AB系统 在H4 ′和H5 ′/H5 ″之间具有小的耦合。 因此，ECOSY光谱 将必须按照“JH '.H5”和3 JH 4'-H5”的顺序进行模拟。 三是 P的化学位移各向异性可能导致相当大的线 加宽，使得难以提取SE小耦合 constants. 关于第一个问题，较高的频率可以 提供一个部分的解决方案;然而，这将加剧第三个 问题. 关于第二个问题，我们研究了 用于HF、H2 '和H2”的典型值的计算机生成的ECOSY光谱 以及用于H4 '、H5'和H5”。 可以看出，对于H2 '/H2”， 七个强度相等的波瓣，其中cc和P自旋状态是 在CO2中充分置换，在（O 1）中刚好重叠。 对于H5 '/H5“， 是八瓣，自旋态的位移是相反的 对于H2 '/H2”观察到的。 这种外观上的差异， 与孪晶耦合的大小有关， J12'512”和J 4' 5 '/4' 5”。 在H5 ′/H5 ″系统中， 外部8个叶片明显小于内部8个叶片 因为它是典型的AB系统。 探讨了影响 化学位移各向异性，我们记录了ECOSY光谱的11聚体 在500、600和750 MHz NMR系统下， 在存在和不存在“P解耦”的情况下的温度。 在 在没有“P”退耦的情况下，质子线宽随着 增加磁场强度。 “另一方面， 导致了ECOSY光谱的显著改善，其中包含 8个肺叶的多联体显示典型的AB模式。 因此我们 已经表明，“P解耦ECOSY光谱可以用于 明确地提取JH 4 '-H5'和JH 4 '-H5”偶合常数， DNA寡核苷酸。 然而，该方法仅适用于 那些显示其H4 '-H5'/H5”交叉峰远离 对角线。 不幸的是，这些结论是基于“P解耦 600 MHz下的ECOSY实验。 我们尝试执行“P解耦 在750 Mhz的ECOSY导致4 '，5'和5”的信号丢失 地区