Binder Hans, Preibisch Stephan
Interdisciplinary Centre for Bioinformatics, University of Leipzig, Leipzig, Germany.
Biophys J. 2005 Jul;89(1):337-52. doi: 10.1529/biophysj.104.055343. Epub 2005 Apr 15.
Gene expression analysis by means of microarrays is based on the sequence-specific binding of RNA to DNA oligonucleotide probes and its measurement using fluorescent labels. The binding of RNA fragments involving sequences other than the intended target is problematic because it adds a chemical background to the signal, which is not related to the expression degree of the target gene. The article presents a molecular signature of specific and nonspecific hybridization with potential consequences for gene expression analysis. We analyzed the signal intensities of perfect match (PM) and mismatch (MM) probes of GeneChip microarrays to specify the effect of specific and nonspecific hybridization. We found that these events give rise to different relations between the PM and MM intensities as function of the middle base of the PM, namely a triplet-like (C > G approximately T > A > 0) and a duplet-like (C approximately T > 0 > G approximately A) pattern of the PM-MM log-intensity difference upon binding of specific and nonspecific RNA fragments, respectively. The systematic behavior of the intensity difference can be rationalized on the level of basepairings of DNA/RNA oligonucleotide duplexes in the middle of the probe sequence. Nonspecific binding is characterized by the reversal of the central Watson-Crick (WC) pairing for each PM/MM probe pair, whereas specific binding refers to the combination of a WC and a self-complementary (SC) pairing in PM and MM probes, respectively. The Gibbs free energy contribution of WC pairs to duplex stability is asymmetric for purines and pyrimidines of the PM and decreases according to C > G approximately T > A. SC pairings on the average only weakly contribute to duplex stability. The intensity of complementary MM introduces a systematic source of variation which decreases the precision of expression measures based on the MM intensities.
通过微阵列进行基因表达分析是基于RNA与DNA寡核苷酸探针的序列特异性结合，并使用荧光标记进行测量。涉及预期靶标以外序列的RNA片段的结合存在问题，因为它会给信号增加化学背景，而这与靶基因的表达程度无关。本文提出了特异性和非特异性杂交的分子特征及其对基因表达分析的潜在影响。我们分析了基因芯片微阵列中完全匹配（PM）和错配（MM）探针的信号强度，以明确特异性和非特异性杂交的影响。我们发现，这些事件会导致PM和MM强度之间呈现出不同的关系，该关系是PM中间碱基的函数，即分别在特异性和非特异性RNA片段结合时，PM-MM对数强度差异呈现出类似三联体的模式（C > G ≈ T > A > 0）和类似二联体的模式（C ≈ T > 0 > G ≈ A）。强度差异的系统行为可以在探针序列中间的DNA/RNA寡核苷酸双链体的碱基配对水平上得到合理解释。非特异性结合的特征是每个PM/MM探针配对的中心沃森-克里克（WC）配对发生反转，而特异性结合分别是指PM和MM探针中WC配对和自我互补（SC）配对的组合。WC配对对双链体稳定性的吉布斯自由能贡献对于PM的嘌呤和嘧啶是不对称的，并且按照C > G ≈ T > A的顺序降低。SC配对平均而言对双链体稳定性的贡献较弱。互补MM的强度引入了一个系统的变异来源，这降低了基于MM强度的表达测量的精度。
