Stabilization of Three Stranded Nucleic Acid Structures

早些时候，我们计算了立体化学上可行的DNA三螺旋(所谓的R-型，或重组三链)。最近在用碘-125放射性探针研究的T7转录复合体中发现了一种类似R-型的DNA-RNA结构。在转录“气泡”的“上游”端(聚合酶“裂隙”外)观察到三链的联合DNA-DNA-RNA。这种结构的功能作用可能与伸长转录复合体的稳定有关。这种DNA/RNA结合也可能被修复酶用来识别DNA双链中的错配。我们认为，对于WC对和错配，RNA：DNA+DNA和RNA+DNA：DNA两种状态之间的平衡会不同(在后一种情况下，平衡将向RNA：DNA+DNA移动，第三条DNA链将主要暴露)。为了验证这一假设，我们启动了一个新项目。 具体地说，溶液中和转录延伸复合体中的三个DNA/RNA链结构将用几种光谱方法进行分析。为了促进核内R-三联体的形成和避免聚集，我们根据我们的计算设计了寡核苷酸序列。首先，将只使用DNA结构，稍后还将添加RNA。“封闭”和“开放”状态之间的平衡将通过紫外光、镉和荧光测量进行监测。为此，将使用以蝶啶为基础的荧光核苷类似物6-甲基异黄蝶呤(F)作为探针。我们的数据表明，最稳定的三联体在5‘端有F-探针，其次是胸腺嘧啶。重要的是，这一观察结果与我们早先计算的R型结构是一致的，并且可以通过简单的立体化学考虑来解释。最佳的寡核苷酸将通过核磁共振进行研究。 在第二阶段，我们打算研究DNA链中引入的各种错配的DNA-DNA-RNA三联体，包括实时荧光测量。这些研究将为转录过程中DNA和RNA的开启-关闭转变提供独特的热力学和动力学知识。此外，这些结果将有助于理解实际方面，例如可能涉及与转录相关的错配识别的立体化学机制。 Z01 BC 10046-05

Earlier, we calculated the stereochemically feasible DNA triple helix (the so called R-form, or recombination triplex). A DNA-RNA structure, similar to R-form, was recently detected in the T7 transcription complex studied by iodine-125 radioprobing. The three-stranded associate DNA-DNA-RNA was observed at the "upstream" end of the transcription "bubble" (outside the polymerase "cleft"). The functional role of this structure is likely related to stabilization of the elongation transcription complex. It is also possible that this DNA/RNA association is used by the repair enzymes for recognition of mismatches in the DNA duplex. We suggest that the equilibrium between the two states, RNA:DNA+DNA and RNA+DNA:DNA, would be different for the WC pair and a mismatch (in the latter case the equilibrium will be shifted toward RNA:DNA+DNA, and the third DNA strand will be predominantly exposed). To test this hypothesis, we have initiated a new project. Specifically, the three stranded DNA/RNA structures in solution and in transcription elongation complexes are to be analyzed with several spectroscopic methods. To facilitate formation of the intranucleotide R-triplexes and to avoid aggregation, we designed the oligonucleotide sequences based on our calculations. First, only the DNA constructs will be used, and later RNA will be added as well. The equilibrium between the "closed" and the "open" states will be monitored by UV, CD and fluorescence measurements. To this aim, the fluorescent pteridine-based nucleoside analog, 6-methylisoxanthopterin (F), will be used as a probe. Our data indicate that the most stable triplexes would have the F-probe at the 5'-end followed by thymine. Importantly, this observation is consistent with the R-form structure calculated by us earlier, and can be explained by means of simple stereochemical considerations. The optimal oligonucleotides are to be studied by NMR. In a second stage, we intend to study the DNA-DNA-RNA triplexes with various mismatches introduced in the DNA strands, including fluorescence measurements in real time. These studies would provide a unique knowledge of the thermodynamics and kinetics of the opening-closing transitions in DNA and RNA during transcription. In addition, these results would be useful for understanding practical aspects, such as the stereochemical mechanisms likely to be involved in the transcription-related recognition of mismatches. Z01 BC 10046-05