INFLUENCE OF LOOP RESIDUES ON THE RELATIVE STABILITIES OF DNA HAIRPIN STRUCTURES
INFLUENCE OF LOOP RESIDUES ON THE RELATIVE STABILITIES OF DNA HAIRPIN STRUCTURES
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DOI：
10.1073/pnas.85.17.6242
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发表时间：
1988-09-01
影响因子：
11.1
通讯作者：
BRESLAUER, KJ
中科院分区：
文献类型：
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作者：
SENIOR, MM;JONES, RA;BRESLAUER, KJ
我们已经确定了DNA发夹结构的相对稳定性和熔化行为作为环中非键残基的函数。我们在这项工作中研究的特殊发夹结构家族由16-聚体序列d[CGAACG(X)4CGTTCG]组成，其中X是脱氧腺苷、脱氧胞苷、脱氧鸟苷或脱氧胸苷。这种16-聚体可以自我折叠，形成一个DNA发夹结构家族，拥有一个共同的六聚体茎双链和一个由4个核苷酸组成的非结合环。对于本工作中研究的发痛结构，我们改变了环组成，从所有的嘌呤残基到所有的嘧啶残基。我们通过光谱和量热技术的结合，从热力学上表征了这些发夹结构的相对稳定性和熔融轮廓。为了建立热力学“基线”，我们还对分离的六聚体双链d[(CGAACG).cntdo.(CGTTCG)]进行了平行研究，它对应于每个发夹结构中存在的共同茎双链。我们的光谱和量热数据表明：(I)环中含有4个dt残基的发夹结构表现出最高的熔化温度，而相应的环中含有4个da残基的发夹结构表现出最低的熔化温度。(Ii)25度的自由能数据。C揭示了所研究的四种结构的DNA发夹稳定性的顺序：T环>；C环>；G环>；A环。也就是说，四个残基的嘧啶环发夹比嘌呤环发夹更稳定。(Iii)发夹稳定性的环相关顺序与量热法确定的发夹破坏的转变热的类似趋势是平行的。因此，相对于嘌呤环发夹结构而言，嘧啶环发夹结构的稳定性增强在起源上是焓的。为了深入了解热力学差异的分子基础，我们使用质子核磁共振光谱来探索最稳定的发夹结构(T环)和最不稳定的发夹结构(A环)之间的结构差异。二维核Overhauser增强光谱显示了两个发夹结构的茎双链中的残基之间的连接性，这与B-型DNA一致。此外，T环和A环中的非结合残基表现出相同的连接模式。然而，在茎-环连接的5‘’侧，T-环残基表现出与茎双链的相邻碱基对的连通性，这在相应的A-环残基中是没有观察到的。茎-环结连接性的差异可能为我们观察到T环发夹结构比相应的A环发夹结构更稳定提供了结构基础。
We have determined the relative stabilities and melting behaviors of DNA hairpin structures as a function of the nonbonded residues in the loop. The specific family of hairpin structures we investigated in this work is formed by the 16-mer sequence d[CGAACG(X)4CGTTCG], where X is deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine. This 16-mer can fold back on itself to form a family of DNA hairpin structures that possess a common hexameric stem duplex and a nonbonded loop of 4 nucleotides. For the hairpain structures investigated in this work, we varied the loop composition from all purine residues to all pyrimidine residues. We thermodynamically characterized the relative stabilities and melting profiles of these hairpin structures by a combination of spectroscopic and calorimetric techniques. To establish a thermodynamic "baseline," we also conducted parallel studies on the isolated hexameric duplex d[(CGAACG).cntdot.(CGTTCG)], which corresponds to the common stem duplex present in each hairpin structure. Our spectroscopic and calorimetric data reveal the following: (i) The hairpin structure with four dT residues in the loop exhibits the highest melting temperature, while the corresponding hairpin structure with four dA residues in the loop exhibits the lowest melting temperature. (ii) The free energy data at 25.degree. C reveal the following order of DNA hairpin stability for the four structures studied here: T loop > C loop > G loop > A loop. In other words, the pyrimidine-looped hairpins of four residues are more stable than the purine- looped hairpins. (iii) The loop-dependent order of hairpin stability is paralleled by a similar trend in the calorimetrically determined transition enthalpies for hairpin disruption. Thus, the enhanced stability of the pyrimidine-looped hairpin structures relative to purine-looped hairpin structures is enthalpic in origin. To develop insight into the molecular basis for the thermodynamic differences, proton NMR spectroscopy was used to probe for structural disparities between the most stable hairpin structure (T loop) and the least stable hairpin structure (A loop). Two-dimensional nuclear Overhauser enhancement spectroscopy revealed connectivities between the residues in the stem duplexes of both hairpin structures that are consistent with B-form DNA. In addition, the nonbonded residues in both the T and A loops exhibited the same connectivity patterns. However, on the 5'' side of the stem-loop junction, the T-loop residue exhibited a connectivity with the adjacent base pair of the stem duplex that is not observed for the corresponding A-loop residue. The difference in connectivities at the stem-loop junction may provide a structural basis for our observation that the T-looped hairpin structure is more stable than the corresponding A-looped hairpin structure.