Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting.
Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting.
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DOI：
10.1073/pnas.2112496119
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发表时间：
2022-06-14
影响因子：
11.1
通讯作者：
中科院分区：
文献类型：
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作者：
核酸采用非天然构象的热力学偏好对于理解它们的功能至关重要，但事实证明很难通过实验进行测量。因此，人们对这些热力学偏好如何随序列和结构背景、生理条件和化学修饰而变化知之甚少。在这里，我们展示了稳定非天然构象并使其成为主要状态的修饰，结合熔融实验，能够方便地测量热力学偏好以在DNA和RNA中形成各种非天然构象。Delta-Melt为串联Hoogsteen碱基对的形成提供了罕见的见解，并揭示了DNA中形成G-C+和A-T Hoogsteen和A-T碱基开放构象的巨大而独特的序列特异性偏好，这可能有助于序列特异性DNA生物化学。形成非天然构象的热力学偏好对于理解核酸如何折叠和功能至关重要。然而，它们很难在实验上测量，因为这需要准确地确定大量其他构象中少量低丰度(<；10%)构象的数量。在这里，我们展示了熔融实验能够方便地测量热力学偏好，以采用DNA和RNA中的非自然构象。这种“增量-熔融”方法的关键是使用化学修饰来使特定的次要非天然构象成为主要状态。通过使用核磁共振对热力学参数的独立测量，建立了三角洲熔体在不同生理条件和序列背景下对四种不同非天然构象的有效性和稳健性。Delta-MALLET比核磁共振更快，简单，成本效益高，能够测量异常低密度构象的热力学偏好。使用增量熔融，我们获得了罕见的构象协作性的见解，获得了同时形成两个相邻的Hoogsteen碱基对时显著的协作性(1.0至2.5kcal/mol)的证据。我们还测量了几乎所有16个三核苷酸序列背景下形成G-C+和A-T Hoogsteen和A-T碱基开放状态的热力学偏好，发现明显的序列特异性变化在2-3kcal/mol的数量级。DNA双螺旋中这种丰富的序列特异性非天然次要构象可能有助于塑造DNA生物化学的序列特异性。因此，熔融实验现在可以用来获取有关生物分子自由能景观区域的热力学信息，这些区域超出了天然折叠和展开构象。
Thermodynamic preferences of nucleic acids to adopt non-native conformations are crucial for understanding how they function but prove difficult to measure experimentally. As a result, little is known about how these thermodynamic preferences vary with sequence and structural contexts, physiological conditions, and chemical modifications. Here, we show that modifications stabilizing non-native conformations and rendering them the major state, in conjunction with melting experiments, enable facile measurements of thermodynamic preferences to form various nonnative conformations in DNA and RNA. Delta-melt provided rare insights into the cooperativity of forming tandem Hoogsteen base pairs and revealed large and distinct sequence-specific preferences to form G-C+ and A-T Hoogsteen and A-T base open conformations in DNA, which may contribute to sequence-specific DNA biochemistry. Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this “delta-melt” approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.
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影响因子：
64.8
作者：
Bouvignies, Guillaume;Vallurupalli, Pramodh;Hansen, D. Flemming;Correia, Bruno E.;Lange, Oliver;Bah, Alaji;Vernon, Robert M.;Dahlquist, Frederick W.;Baker, David;Kay, Lewis E.
通讯作者：
Kay, Lewis E.
宇宙（癌症中的体细胞突变目录）数据库和网站。
影响因子：
8.8
作者：
Bamford, S;Dawson, E;Forbes, S;Clements, J;Pettett, R;Dogan, A;Flanagan, A;Teague, J;Futreal, PA;Stratton, MR;Wooster, R
通讯作者：
Wooster, R
影响因子：
46.9
作者：
Berger, Michael F.;Philippakis, Anthony A.;Bulyk, Martha L.
通讯作者：
Bulyk, Martha L.
DOI：
10.1073/pnas.052675499
发表时间：
2002-03-05
影响因子：
11.1
作者：
Abrescia, NGA;Thompson, A;Subirana, JA
通讯作者：
Subirana, JA
影响因子：
5.6
作者：
BORER, PN;DENGLER, B;UHLENBECK, OC
通讯作者：
UHLENBECK, OC