Department of Chemistry, Furman University, Greenville, SC 29613, USA.
Biochemistry. 2011 Feb 1;50(4):458-65. doi: 10.1021/bi101756e. Epub 2010 Dec 31.
The etiology of a large class of inherited neurological diseases is founded on hairpin structures adopted by repeated DNA sequences, and this folding is determined by base sequence and DNA context. Using single substitutions of adenine with 2-aminopurine, we show that intrastrand folding in repeated CAG trinucleotides is also determined by the number of repeats. This isomeric analogue has a fluorescence quantum yield that varies strongly with solvent exposure, thereby distinguishing particular DNA motifs. Prior studies demonstrated that (CAG)(8) alone favors a stem-loop hairpin, yet the same sequence adopts an open loop conformation in a three-way junction. This comparison suggests that repeat folding is disrupted by base pairing in the duplex arms and by purine-purine mismatches in the repeat stem. However, these perturbations are overcome in longer CAG repeats, as demonstrated by studies of isolated and integrated forms of (CAG)(15). The oligonucleotide alone forms a symmetrically folded hairpin with looplike properties exhibited by the relatively high emission intensities from a modification in the central eighth repeat and with stemlike properties evident from the relatively low emission intensities from peripheral modifications. Significantly, these hairpin properties are retained when (CAG)(15) is integrated into a duplex. Intrastrand folding by (CAG)(15) in the three-way junction contrasts with the open loop adopted by (CAG)(8) in the analogous context. This distinction suggests that cooperative interactions in longer repeat tracts overwhelm perturbations to reassert the natural folding propensity. Given that anomalously long repeats are the genetic basis of a large class of inherited neurological diseases, studies with (CAG)-based three-way junctions suggest that their secondary structure is a key factor in the length-dependent manifestation and progression of such diseases.
一类大量遗传性神经疾病的病因是由重复 DNA 序列形成的发夹结构，这种折叠由碱基序列和 DNA 环境决定。我们使用腺嘌呤的单取代 2-氨基嘌呤，证明了重复 CAG 三核苷酸的链内折叠也由重复次数决定。这种异构体类似物的荧光量子产率随溶剂暴露而强烈变化，从而区分特定的 DNA 基序。先前的研究表明，（CAG）8 单独有利于茎环发夹结构，但相同的序列在三链结中采用开环构象。这种比较表明，重复折叠被双链臂中的碱基配对和重复茎中的嘌呤-嘌呤错配破坏。然而，这些干扰在更长的 CAG 重复中被克服，这可以通过对分离和整合形式的（CAG）15 的研究来证明。寡核苷酸本身形成对称折叠的发夹，其具有Loop 样性质，表现为中央第八个重复修饰的相对高发射强度，并且具有 Stem 样性质，表现为外围修饰的相对低发射强度。重要的是，当（CAG）15 整合到双链中时，这些发夹性质得以保留。（CAG）15 在三链结中的链内折叠与（CAG）8 在类似环境中采用的开环形成对比。这种区别表明，在较长重复片段中的协同相互作用克服了对重新建立自然折叠倾向的干扰。鉴于异常长的重复是一大类遗传性神经疾病的遗传基础，基于（CAG）的三链结研究表明，它们的二级结构是这些疾病长度依赖性表现和进展的关键因素。
