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Impact of a Snap-Back Loop on Stability and Ligand Binding to a Parallel G-Quadruplex
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2020-03-25 , DOI: 10.1021/acs.jpcb.0c00700 Lena Schnarr 1 , Jagannath Jana 1 , Pit Preckwinkel 1 , Klaus Weisz 1
The Journal of Physical Chemistry B ( IF 2.8 ) Pub Date : 2020-03-25 , DOI: 10.1021/acs.jpcb.0c00700 Lena Schnarr 1 , Jagannath Jana 1 , Pit Preckwinkel 1 , Klaus Weisz 1
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Various genomic DNA sequences including a MYC promoter sequence are amenable to the formation of a G-quadruplex featuring a snap-back loop with the incorporation of a 3′-terminal guanine into the quadruplex core. To evaluate relative stabilities and ligand binding in more detail, optical, microcalorimetric, and NMR structural studies were performed on both a minimal mutant sequence Pu22T that exclusively folds into a snap-back loop quadruplex and a parallel MYC quadruplex proposed to be the most relevant fold of the MYC promoter in a cellular environment. Similar thermal stabilities for Pu22T and MYC suggest the coexistence of both quadruplexes when derived from a sequence able to fold into both topologies. Isothermal titration calorimetry indicates a mostly identical enthalpy-driven strong binding of an indoloquinoline ligand but with a reduced number of high-affinity binding sites in Pu22T in line with a novel modified FRET competitive melting assay. Corroborated by fluorescence titrations using 2-aminopurine as a fluorescent probe, NMR chemical shift footprints show binding of the ligand at the Pu22T 5′-outer tetrad with the formation of a binding pocket. On the other hand, steric restrictions due to the snap-back loop severely restrict ligand stacking on the 3′-outer tetrad of Pu22T.
中文翻译：
Snap-Back循环对稳定性和配体与平行G-四链体结合的影响
包括MYC启动子序列在内的各种基因组DNA序列适合于形成G-四链体，该G-四链体具有将3'-末端鸟嘌呤掺入到四链体核心中的回扣环。为了更详细地评估相对稳定性和配体结合，对最小折叠序列Pu22T进行了光学，微量量热和NMR结构研究，该序列仅折叠成折回环四倍体，而平行MYC四倍体被认为是最相关的折叠在细胞环境中的MYC启动子。Pu22T和MYC具有相似的热稳定性当从能够折叠成两种拓扑结构的序列中衍生出来时，表明两种四链体并存。等温滴定量热法表明，吲哚喹啉配体具有几乎相同的焓驱动强烈结合，但Pu22T中高亲和力结合位点数量减少，这与新型改良的FRET竞争熔解法一致。使用2-氨基嘌呤作为荧光探针的荧光滴定证实了这一点，NMR化学位移足迹显示了配体在Pu22T 5'-外四联体处的结合，并形成了一个结合袋。另一方面，归因于快速回环的空间限制严重限制了配体在Pu22T的3'-外四分体上的堆积。
更新日期：2020-03-26
中文翻译：
Snap-Back循环对稳定性和配体与平行G-四链体结合的影响
包括MYC启动子序列在内的各种基因组DNA序列适合于形成G-四链体，该G-四链体具有将3'-末端鸟嘌呤掺入到四链体核心中的回扣环。为了更详细地评估相对稳定性和配体结合，对最小折叠序列Pu22T进行了光学，微量量热和NMR结构研究，该序列仅折叠成折回环四倍体，而平行MYC四倍体被认为是最相关的折叠在细胞环境中的MYC启动子。Pu22T和MYC具有相似的热稳定性当从能够折叠成两种拓扑结构的序列中衍生出来时，表明两种四链体并存。等温滴定量热法表明，吲哚喹啉配体具有几乎相同的焓驱动强烈结合，但Pu22T中高亲和力结合位点数量减少，这与新型改良的FRET竞争熔解法一致。使用2-氨基嘌呤作为荧光探针的荧光滴定证实了这一点，NMR化学位移足迹显示了配体在Pu22T 5'-外四联体处的结合，并形成了一个结合袋。另一方面，归因于快速回环的空间限制严重限制了配体在Pu22T的3'-外四分体上的堆积。
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