Department of Physics , Loyola University Chicago , Chicago , Illinois 60660 , United States.
J Phys Chem B. 2018 Dec 20;122(50):11841-11851. doi: 10.1021/acs.jpcb.8b07994. Epub 2018 Dec 3.
Repetitive trinucleotide DNA sequences at specific genetic loci are associated with numerous hereditary, neurodegenerative diseases. The propensity of single-stranded domains containing these sequences to form secondary structure via extensive self-complementarity disrupts normal DNA processing to create genetic instabilities. To investigate these intrastrand structural dynamics, a DNA hairpin system was devised for single-molecule fluorescence study of the folding kinetics and energetics for secondary structure formation between two interacting, repetitive domains with specific numbers of the same trinucleotide motif (CXG), where X = T or A. Single-molecule fluorescence resonance energy transfer (smFRET) data show discrete conformational transitions between unstructured and closed hairpin states. The lifetimes of the closed hairpin states correlate with the number of repeats, with (CTG) /(CTG) domains maintaining longer-lived, closed states than equivalent-sized (CAG) /(CAG) domains. NaCl promotes similar degree of stabilization for the closed hairpin states of both repeat sequences. Temperature-based, smFRET experiments reveal that NaCl favors hairpin closing for (CAG) /(CAG) by preordering single-stranded repeat domains to accelerate the closing transition. In contrast, NaCl slows the opening transition of CTG hairpins; however, it promotes misfolded conformations that require unfolding. Energy diagrams illustrate the distinct folding pathways of (CTG) and (CAG) repeat domains and identify features that may contribute to their gene-destabilizing effects.
特定遗传位置的重复三核苷酸 DNA 序列与许多遗传性、神经退行性疾病有关。这些序列中含有单链结构域的倾向，通过广泛的自我互补形成二级结构，破坏了正常的 DNA 加工，从而导致遗传不稳定性。为了研究这些链内结构动力学，设计了一种 DNA 发夹系统，用于对两个相互作用的重复结构域之间的二级结构形成的折叠动力学和热力学进行单分子荧光研究，这两个重复结构域具有相同的三核苷酸基序 (CXG) 的特定数量，其中 X = T 或 A。单分子荧光共振能量转移 (smFRET) 数据显示了未结构化和闭合发夹状态之间的离散构象转变。闭合发夹状态的寿命与重复次数相关，（CTG）/（CTG） 结构域比等效大小的（CAG）/（CAG） 结构域具有更长寿命的闭合状态。NaCl 对两种重复序列的闭合发夹状态都具有相似程度的稳定作用。基于温度的 smFRET 实验表明，NaCl 通过预组织单链重复结构域来加速闭合转变，从而有利于（CAG）/（CAG） 的发夹闭合。相比之下，NaCl 会减缓 CTG 发夹的打开转变；然而，它会促进需要展开的错误折叠构象。能量图说明了（CTG）和（CAG）重复结构域的独特折叠途径，并确定了可能导致它们基因不稳定效应的特征。
