粘性末端短链DNA环化-脱环反应动力学的影响因素
Determinants of cyclization-decyclization kinetics of short DNA with sticky ends
摘要：
具有粘性末端的DNA的环化通常用于构建DNA微环和测量DNA可弯曲性。短DNA（< 150 bp）的环化概率具有很强的长度依赖性，但它如何依赖于粘性末端围绕螺旋轴的旋转定位尚不清楚。为了阐明短DNA环化概率的决定因素，我们使用单分子荧光共振能量转移（FRET）测量了具有粘性末端的~100-bp DNA在两个螺旋周期内的环化和解环速率。环化速率随长度单调增加，表明没有过度扭曲，而脱环速率随长度振荡，在半整数螺旋圈处较高，在整数螺旋圈处较低。振荡曲线在动力学和动力学上与三态环化模型一致，其中粘性末端的短DNA首先弯曲成扭转松弛的泪滴，随后在末端碱基堆叠时过渡到更稳定的环。我们还表明，从这项研究中提取的成环概率密度（J因子）与蠕虫状链模型在100 bp附近是很好的协议。对于较短的DNA，我们讨论了各种实验因素，防止J因子的准确测量。
英文摘要：
Cyclization of DNA with sticky ends is commonly used to construct DNA minicircles and to measure DNA bendability. The cyclization probability of short DNA (< 150 bp) has a strong length dependence, but how it depends on the rotational positioning of the sticky ends around the helical axis is less clear. To shed light upon the determinants of the cyclization probability of short DNA, we measured cyclization and decyclization rates of ~100-bp DNA with sticky ends over two helical periods using single-molecule Fluorescence Resonance Energy Transfer (FRET). The cyclization rate increases monotonically with length, indicating no excess twisting, while the decyclization rate oscillates with length, higher at half-integer helical turns and lower at integer helical turns. The oscillation profile is kinetically and thermodynamically consistent with a three-state cyclization model in which sticky-ended short DNA first bends into a torsionally-relaxed teardrop, and subsequently transitions to a more stable loop upon terminal base stacking. We also show that the looping probability density (the J factor) extracted from this study is in good agreement with the worm-like chain model near 100 bp. For shorter DNA, we discuss various experimental factors that prevent an accurate measurement of the J factor.
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