Yilmaz L Safak, Noguera Daniel R
Department of Civil and Environmental Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, Wisconsin 53706-1691, USA.
Biotechnol Bioeng. 2007 Feb 1;96(2):349-63. doi: 10.1002/bit.21114.
Stringency in ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH) is typically adjusted with formamide, and the optimum formamide concentration at which the probe can hybridize with the target rRNA, but not with rRNAs with mismatches, is to be found experimentally. This is a difficult task when target or closest non-target organisms are not available in pure culture, or when there are numerous non-targets of concern. The objective of this work was to formulate mechanistic models capable of simulating the effect of formamide on probe dissociation. Using a previously described equilibrium model of FISH [Yilmaz and Noguera (2004) Applied and Environmental Microbiology 70(12):7126-7139] as the basis, the effect of formamide on free energy changes of probe-target duplex formation (DeltaG(1)(0)) and folding of target region (DeltaG(3)(0)) was simulated to be linear, and models differing in the definitions of the slopes of these relationships (m(1) and m(3)) were calibrated using experimental dissociation profiles for 27 probes targeting the 16S rRNA of Escherichia coli (E. coli). A good level of predictive power was obtained when m(1) was linearly related to probe length and when m(3) was made proportional to DeltaG(3)(0). The effect of single mismatches on probe dissociation with formamide was also studied, although at a preliminary level. The expected changes in DeltaG(1)(0) with the introduction of mismatches were not sufficient to capture the overall trends of mismatched dissociation profiles. In conclusion, this study offers the first theoretical method to calculate dissociation profiles for perfectly matched probes, and suggests a direction to systematically evaluate the effect of formamide on mismatched probes.
针对核糖体RNA（rRNA）的荧光原位杂交（FISH）的严谨性通常用甲酰胺来调节，而能使探针与目标rRNA杂交但不与错配rRNA杂交的最佳甲酰胺浓度需要通过实验来确定。当目标生物或最接近的非目标生物无法进行纯培养，或者存在众多需要关注的非目标生物时，这是一项艰巨的任务。这项工作的目的是建立能够模拟甲酰胺对探针解离影响的机理模型。以先前描述的FISH平衡模型[Yilmaz和Noguera（2004年），《应用与环境微生物学》70(12):7126 - 7139]为基础，模拟了甲酰胺对探针 - 靶标双链体形成的自由能变化（ΔG(1)(0)）和靶标区域折叠的自由能变化（ΔG(3)(0)）的影响是线性的，并使用针对大肠杆菌（E. coli）16S rRNA的27个探针的实验解离图谱，对这些关系斜率（m(1)和m(3)）定义不同的模型进行了校准。当m(1)与探针长度呈线性相关且m(3)与ΔG(3)(0)成比例时，获得了良好的预测能力。尽管只是初步研究，但也研究了单个错配对甲酰胺介导的探针解离的影响。引入错配时预期的ΔG(1)(0)变化不足以捕捉错配解离图谱的总体趋势。总之，本研究提供了第一种计算完全匹配探针解离图谱的理论方法，并为系统评估甲酰胺对错配探针的影响指明了方向。
