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: 6355177
• 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: 2000
• 资助国家: 美国
• 起止时间: 2000-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6355177
• 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中环外C4'-C5'键的构象 骨干在DNA的弯曲过程中起着至关重要的作用。 构象者 关于该键可以表示为快速平衡混合物 三种经典旋转异构体：gauche' (g')、gauche- (g-) 和 trans （t）。 例如，从gauche+构象异构体到反式构象异构体的转变 将增加残基之间的磷酸间距离。 这将 引起局部拆垛，从而使碱基对滚入 主要凹槽，形成扭结或铰链。 在晶体中， 压倒性优选的构象是 g+ 和一些 t， g- 很少被观察到。 检查 P-P 距离 vis-a vis 17 个 B-DNA、32 个 A-DNA 和 11 个中的 C5'-C4' 扭转 分辨率优于 2.5A 的 RNA 晶体结构揭示了 以下：g' 出现 652 次，反式构象出现 54 次 C5'-C4'键。 反式构象中的 P-P 距离始终为 发现比 g'conformer 中的要大。 然而， 尝试通过高通量确定 DNA 弯曲的序列依赖性 在没有蛋白质的情况下 DNA 双链体的分辨率晶体学， 通常会受到与晶体堆积相关的伪影的阻碍。 这 开发了ECOSY技术来直接提取小型无源 存在大型主动联轴器的联轴器。 因为H5'/H5" 是一个孪生系统，应该可以使用 ECOSY 提取“JH”-H5 值的技术。 和‘JHI’-H5”。然而， 这种方法可能存在三个问题。 第一的， 114' 和 H5'/H5" 之间的许多 ECOSY 交叉峰预计会 保持非常接近对角线。 二、H2'的化学位移 脱氧核糖环的H2”和H2”通常显着不同 彼此远离并且远离相应的Hl'；这 JHI'-H2'和JHI'-H2"的星等很大，在6到9之间 赫兹。另一方面，H4'-H5'/H5"是一个紧耦合的AB系统 H4' 和 H5'/H5" 之间的耦合很小。因此，ECOSY 谱 必须按“JH”.H5' 和 3 JH4'-H5" 的顺序进行模拟。第三， “P 的化学位移各向异性可能会导致相当大的线 展宽，使得提取这些小的耦合变得困难 常数。 对于第一个问题，更高的频率可能 提供部分解决方案；但这会加剧第三种情况 问题。 针对第二个问题，我们研究了 计算机生成的 H F、H2'& H2 典型值 ECOSY 谱图" 以及 H4'、H5' 和 H5"。可以看出，对于 H2'/H2"，有 七个等强度的波瓣，其中 cc 和 P 自旋态为 在 co2 中充分置换并且在 (o 1. 对于 H5'/H5 " 中重叠 是八个波瓣，自旋态的位移相反 与 H2'/H2" 观察到的结果相同。这种外观差异 与孪生耦合的大小有关 J12'512" 和 J4'5'/4'5"。 在 H5'/H5" 系统中， 外部8个叶明显小于内部8个叶 因为它是典型的AB系统。 为了调查效果 化学位移各向异性我们记录了 11 mer 的 ECOSY 光谱 500、600 和 750 MHz NMR 系统的双工 AAA 在存在和不存在“P 解耦的情况下的温度。在 没有“P 解耦，质子线宽随着 增加磁场强度。 “另一方面，P 解耦， 导致 ECOSY 光谱显着改善，其中包含 具有 8 个瓣的多重峰，显示典型的 AB 模式。 因此，我们 已经表明“P 解耦 ECOSY 谱可用于 明确提取 JH4'-H5' 和 JH4'-H5" 耦合常数 DNA寡核苷酸。 然而，该方法仅适用于 那些显示其 H4'-H5'/H5" 交叉峰的残基 对角线。 不幸的是，这些结论是基于“P解耦 ECOSY 实验在 600 MHz 下进行。 我们尝试执行“P解耦 750 Mhz 的 ECOSY 导致 4'、5' 和 5" 信号丢失 地区。