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

DNA中外环C4‘-C5’键的构象 脊椎在DNA的弯曲过程中起着至关重要的作用。整合者 关于这个键可以表示为一个快速平衡的混合物 三个经典的旋转异构体，guche‘(g’)，guche-(g-)和trans (T)。例如，从Guche+构象到TRANS 会增加残基之间的磷间距离。这将是 导致局部去堆积，从而使碱基对滚动到 主要凹槽，形成扭结或铰链。在晶体中， 压倒性的优选构象是具有少数t的g+， G-很少观察到。P-P距离相对于A-V的检验 17个B-DNA、32个A-DNA和11个A-DNA的C5‘-C4’扭转 分辨率优于2.5A的RNA晶体结构揭示了 如下：g‘出现652次，反式构象54次 C5‘-C4’键。反式构象中的P-P距离总是 发现比G‘Conformator中的要大。然而， 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双链的分辨结晶学， 经常受到与水晶包装相关的人工制品的阻碍。这个 发展了ECOSY技术来直接提取小无源 在大型主动联轴器存在的情况下的联轴器。因为H5‘/H5“ 是一个宝石系统，应该有可能采用经社理事会 技术来提取‘JH’-H5的值。和‘JHI’-H5“。然而， 这种方法可能存在三个问题。第一, 在114‘和H5’/H5“之间的许多ECOSY交叉峰预计将 保持离对角线很近。第二，H_2‘的化学位移 和脱氧核糖环的H2“通常显著不同 并远离相应的hl‘； JHI‘-H2’和JHI‘-H2“的震级很大，在6到9的范围内 赫兹。另一方面，H4‘-H5’/H5“是一个紧耦合的AB系统 具有H4‘和H5’/H5“之间的小耦合。因此，ECOSY光谱 将不得不按顺序‘JH’.H5‘和3’JH4‘-H5“进行模拟。第三， P的化学位移各向异性可能导致相当大的线 加宽，使得提取Se小耦合变得困难 常量。关于第一个问题，更高的频率可能 提供部分解决方案；然而，这将加剧第三个问题 有问题。关于第二个问题，我们已经研究了 H、F、H_2‘和H_2的计算机生成的ECOSY谱“ 以及对于H4‘、H5’和H5“。可以看到，对于H2‘/H2”，存在 七个等强度的波瓣，其中cc和p自旋态是 在二氧化碳中被很好地置换并且仅重叠在(o 1.对于H5‘/H5“ 是八个波瓣，而自旋态的位移是相反的 到观察到的H2‘/H2“。这种外观上的差异 与宝石联结的大小有关 J12‘512“及J4’5‘/4’5”。在H5‘/H5“系统中， 外8叶明显少于内8叶 因为它是一个典型的AB系统。为了调查……的影响 化学位移各向异性我们记录了11mer的ECOSY光谱 500、600和750 MHz核磁共振系统上的双工AAA 在温度存在和不存在“P去耦合”的情况下 在没有“P去耦合”的情况下，质子线宽随 增加磁场强度。“另一方面，P脱钩， 导致ECOSY光谱的显著改善，其中包括 具有8个叶的多重集显示出典型的AB图案。因此，我们 已经证明“P-去耦合ECOSY谱可以用来 明确提取JH4‘-H5’和JH4‘-H5“中的耦合常数 DNA寡核苷酸。但是，该方法仅适用于 那些显示其H4‘-H5’/H5“的残基在 对角线。不幸的是，这些结论是建立在“P脱钩”的基础上的 ECOSY实验频率为600 MHz。我们尝试执行“P脱钩” 在750兆赫时的ECOSY导致4‘、5’和5“的信号丢失 区域。

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.