Trapp Christian, Schenkelberger Marc, Ott Albrecht
Experimentalphysik, Universität des Saarlandes, D-66041 Saarbrücken, Germany.
BMC Biophys. 2011 Dec 13;4:20. doi: 10.1186/2046-1682-4-20.
DNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays.
We synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account.
We show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well.
DNA是生物信息的载体。杂交过程，即具有互补序列的单链形成DNA双螺旋，对所有活细胞都很重要。DNA微阵列与其他生物技术如PCR一样，依赖于DNA杂交。然而，迄今为止，杂交的热力学仅得到部分理解。在这里，我们通过实验和理论方法研究长度不等的寡核苷酸链的杂交，这些链在杂交时会形成一个凸起环。在我们的研究中，我们使用内部合成的DNA微阵列。
我们在探针序列基序中合成了带有额外胸腺嘧啶碱基的微阵列，以便在靶标杂交时出现凸起环。我们观察到杂交链的荧光信号随着凸起环长度的增加而单调下降。这对应于在一到十三个碱基的考虑环长度内双链结合亲和力的降低。通过改变凸起环沿DNA双链的位置，我们观察到相对于链中心的对称信号变化。我们使用热平衡下的分子拉链模型很好地再现了实验结果。然而，需要考虑在凸起环位置通过双链打开而出现的两条链之间的结合状态。
我们表明，具有凸起环的稳定DNA双链可以由长度不等的短链形成，并且它们对微阵列上杂交链的荧光强度有很大贡献。为了借助平衡热力学再现结果至关重要（并且在很好的近似下足够）的是不仅要考虑双链在末端的打开，还要考虑在凸起环位置的打开。尽管本研究中使用的热力学参数取自溶液中的杂交实验，但这些参数与我们的DNA微阵列数据拟合得很好。
