ACID MULTIMERS OF OLIGODEOXYCYTIDINE STRANDS - STOICHIOMETRY, BASE-PAIR CHARACTERIZATION, AND PROTON-EXCHANGE PROPERTIES
ACID MULTIMERS OF OLIGODEOXYCYTIDINE STRANDS - STOICHIOMETRY, BASE-PAIR CHARACTERIZATION, AND PROTON-EXCHANGE PROPERTIES
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DOI：
10.1021/bi00074a013
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发表时间：
1993-06-15
期刊：
影响因子：
2.9
通讯作者：
GUERON, M
中科院分区：
文献类型：
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作者：
LEROY, JL;GEHRING, K;GUERON, M
最近提出的寡核苷酸 5'-d(TC5) 的酸形式的结构是一个四链“四分体”，其中两个平行链、碱基配对的双链体紧密相连，其半质子化的 C-C+ 碱基对面对面且完全插入，形成所谓的“i-motif”（Gehring 等，1993）。我们使用氨基和亚氨基质子光谱来建立碱基对的结构和对称性，这些特性是解析上述酸形式的主要元素。氨基质子谱给出了亚氨基质子跳跃过程速率的最佳下限 (8 x 10(4) s-1)，亚氨基质子跳跃过程负责碱基对对称性。其他脱氧胞苷序列的酸形式的化学计量通过凝胶过滤色谱法进行研究，并且在一种情况下通过NMR平衡滴定进行研究。在所有情况下，即 d(C-12)、d(T2C8T2)、d(C4TC4)、d(TC5)、d(C5)、d(C4)、d(TC4)、d(TC3T) 和 d(TC3)，酸形式以四聚体形式洗脱。在某些情况下还存在单链成分。但除了一些通过高温淬火制备的样品外，没有观察到二聚体。 d(TC5) 结构的特征性 H1'-H1' 残基间 NOESY 交叉峰（Gehring 等人，1993）也在所有已搜索的四聚体中发现，即 d(T2C8T2)、d(C4TC4)、d(TC3T) 和 d(TC3)（未显示），表明这些四聚体也是建立在i-基序并且这种结构通常可以由含有一段少至三个脱氧胞苷的链形成。从 d(TC3) 的 NMR 滴定中，我们得出每个胞苷碱基对从单链形成四聚体的自由能为 -7.6 kJ/mol。将碱基对包装到 i-基序中释放的自由能与形成碱基对本身释放的自由能相当。由于有效的内在交换催化，亚氨基质子交换受到碱基对开放的限制：这解释了添加催化剂缺乏效果的原因。碱基对的寿命比任何 DNA 双链体都要长数百倍，这可能是由于碱基对嵌入的几何形状所致。 d(TC5) 四聚体序列的寿命变化为最近提出的结构提供了支持。内部氨基质子从 C.C+ 对的开放状态进行交换，其速率与适合 C+ 的 pK 9 兼容。但外部质子从封闭状态进行交换，pK 为 17！当有机酸从水溶剂转移到有机溶剂时，会发生类似的 pK 变化。我们还表明，内部氨基质子的交换是开放限制的，尽管它比亚氨基质子交换慢八倍。开放状态的寿命至少为 1 mu，比双工长 100 倍。
The structure recently proposed for the acid form of the oligonucleotide 5'-d(TC5) is a four-strand ''tetrad'' in which two parallel-stranded, base-paired duplexes are intimately associated, with their hemiprotonated C-C+ base pairs face-to-face and fully intercalated, in a so-called ''i-motif'' (Gehring et al., 1993). We use the amino and imino proton spectra to establish the structure and symmetry of the base pairs, properties which are a primary element in the resolution of the acid form described above. The amino proton spectrum gives the best lower limit (8 x 10(4) s-1) on the rate of the imino proton jumping process which is responsible for the base-pair symmetry. The stoichiometry of the acid form of other deoxycytidine sequences is studied by gel filtration chromatography and in one case by an NMR equilibrium titration. In all cases, i.e., d(C-12), d(T2C8T2), d(C4TC4), d(TC5), d(C5), d(C4), d(TC4), d(TC3T), and d(TC3), the acid form elutes as a tetramer. A single-strand component is also present in some cases. But no dimer is observed, except for some samples prepared by quenching from high temperatures. The characteristic H1'-H1' interresidue NOESY cross-peaks of the d(TC5) structure (Gehring et al., 1993) are also found in all the tetramers where they have been searched for, i.e., those of d(T2C8T2), d(C4TC4), d(TC3T), and d(TC3) (not shown), suggesting that these tetramers also are built on the i-motif and that such structures may be formed generally by strands containing a stretch of as little as three deoxycytidines. From the NMR titration of d(TC3), we derive a free energy of -7.6 kJ/mol per cytidine base pair for the formation of the tetramer from single strands. The free energy released by packing a base pair into the i-motif is comparable to that released in forming the base pair itself. Imino proton exchange is limited by base-pair opening, thanks to efficient intrinsic exchange catalysis: this explains the lack of effect of added catalysts. The base-pair lifetime is hundreds of times longer than in any DNA duplex, presumably due to the base-pair intercalation geometry. The variation of the lifetime along the sequence of the d(TC5) tetramer provides support for the recently proposed structure. The internal amino proton exchanges from the open state of the C.C+ pair, at a rate compatible with a pK of 9 appropriate for C+. But the external proton exchanges from the closed state, as with a pK of 17! Comparable pK shifts occur upon transfer of organic acids from an aqueous to an organic solvent. We also show that exchange of the internal amino proton is opening-limited, even though it is eight times slower than imino proton exchange. The lifetime of the open state is at least 1 mus, 100 times longer than in duplexes.