Mandell Kathleen E, Vallone Peter M, Owczarzy Richard, Riccelli Peter V, Benight Albert S
Department of Chemistry, University of Illinois at Chicago, 845 West Taylor Street, Room 4500, 60607, USA.
Biopolymers. 2006 Jun 15;82(3):199-221. doi: 10.1002/bip.20425.
Melting curves and circular dichroism spectra were measured for a number of DNA dumbbell and linear molecules containing dinucleotide repeat sequences of different lengths. To study effects of different sequences on the melting and spectroscopic properties, six DNA dumbbells whose stems contain the central sequences (AA)(10), (AC)(10), (AG)(10), (AT)(10), (GC)(10), and (GG)(10) were prepared. These represent the minimal set of 10 possible dinucleotide repeats. To study effects of dinucleotide repeat length, dumbbells with the central sequences (AG)(n), n = 5 and 20, were prepared. Control molecules, dumbbells with a random central sequence, (RN)(n), n = 5, 10, and 20, were also prepared. The central sequence of each dumbbell was flanked on both sides by the same 12 base pairs and T(4) end-loops. Melting curves were measured by optical absorbance and differential scanning calorimetry in solvents containing 25, 55, 85, and 115 mM Na(+). CD spectra were collected from 20 to 45 degrees C and [Na(+)] from 25 to 115 mM. The spectral database did not reveal any apparent temperature dependence in the pretransition region. Analysis of the melting thermodynamics evaluated as a function of Na(+) provided a means for quantitatively estimating the counterion release with melting for the different sequences. Results show a very definite sequence dependence, indicating the salt-dependent properties of duplex DNA are also sequence dependent. Linear DNA molecules containing the (AG)(n) and (RN)(n), sequences, n = 5, 10, 20, and 30, were also prepared and studied. The linear DNA molecules had the exact sequences of the dumbbell stems. That is, the central repeat sequence in each linear duplex was flanked on both sides by the same 12-bp sequence. Melting and CD studies were also performed on the linear DNA molecules. Comparison of results obtained for the same sequences in dumbbell and linear molecular environments reveals several interesting features of the interplay between sequence-dependent structural variability, sequence length, and the unconstrained (linear) or constrained (dumbbell) molecular environments.
我们测量了许多含有不同长度二核苷酸重复序列的DNA哑铃状分子和线性分子的熔解曲线及圆二色光谱。为了研究不同序列对熔解和光谱性质的影响，我们制备了六种DNA哑铃状分子，其茎部包含中心序列(AA)(10)、(AC)(10)、(AG)(10)、(AT)(10)、(GC)(10)和(GG)(10)。这些代表了10种可能的二核苷酸重复序列的最小集合。为了研究二核苷酸重复长度的影响，我们制备了中心序列为(AG)(n)（n = 5和20）的哑铃状分子。还制备了对照分子，即中心序列为随机序列(RN)(n)（n = 5、10和20）的哑铃状分子。每个哑铃状分子的中心序列两侧均由相同的12个碱基对和T(4)端环组成。在含有25、55、85和115 mM Na⁺的溶剂中，通过光吸收和差示扫描量热法测量熔解曲线。在20至45℃以及[Na⁺]从25至115 mM的条件下收集圆二色光谱。光谱数据库未显示在预转变区域有任何明显的温度依赖性。作为Na⁺的函数对熔解热力学进行分析，为定量估计不同序列熔解时的抗衡离子释放提供了一种方法。结果显示出非常明确的序列依赖性，表明双链DNA的盐依赖性性质也是序列依赖性的。我们还制备并研究了含有(AG)(n)和(RN)(n)序列（n = 5、10、20和30）的线性DNA分子。线性DNA分子具有与哑铃状分子茎部完全相同的序列。也就是说，每个线性双链体中的中心重复序列两侧均由相同的12个碱基对序列组成。我们也对线性DNA分子进行了熔解和圆二色研究。比较在哑铃状分子和线性分子环境中相同序列所获得的结果，揭示了序列依赖性结构变异性、序列长度以及无约束（线性）或受约束（哑铃状）分子环境之间相互作用的几个有趣特征。
