Synthesis of the novel thiophenyl carbazole phosphoramidite DNA building block 5 was accomplished in four steps using a Suzuki–Miyaura cross-coupling reaction from the core carbazole and it was seamlessly accommodated into a 9-mer DNA-based oligonucleotide by incorporation at the flanking 5′-end in combination with a central insertion of an LNA-T nucleotide. The carbazole-containing oligonucleotide was combined in different duplex hybrids, which were characterized by thermal denaturation, circular dichroism and fluorescence studies. The carbazole monomer modulates the duplex stability in various ways. Thus, monomer Z increased the thermal stability of the 9-mer towards the complementary 9-mer/15-mer DNA duplex by 4.2 °C. Furthermore, indications of its intercalation into the duplex were obtained by modeling studies and robust decreases in fluorescence emission intensities upon duplex formation. In contrast, no clear intercalating tendency was corroborated for monomer Z within the DNA/RNA hybrid duplex as indicated by moderate quenching of the fluorescence and similar duplex thermal stabilities relative to the corresponding control duplex. The recognition efficiencies of the carbazole modified oligonucleotide toward single nucleotide mismatches were studied with two 15-mer model targets (DNA and RNA). For both systems, mismatches positioned at the juxtaposition of the carbazole monomer showed pronounced deceases in thermal denaturation temperature. Steady-state fluorescence emission studies of all mismatched duplexes with incorporation of Z monomer typically displayed efficient fluorescence quenching.

中文翻译：

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咔唑修饰寡核苷酸：合成、杂交研究和荧光特性。

使用 Suzuki-Miyaura 交叉偶联反应从核心咔唑分四步完成新型噻吩咔唑亚磷酰胺 DNA 结构单元5的合成，并通过在侧翼 5' 处掺入无缝融入基于 9 聚体 DNA 的寡核苷酸-末端与 LNA-T 核苷酸的中心插入结合。将含咔唑的寡核苷酸组合成不同的双链杂合体，通过热变性、圆二色性和荧光研究对其进行表征。咔唑单体以多种方式调节双链体稳定性。因此，单体Z将 9-mer 对互补的 9-mer/15-mer DNA 双链体的热稳定性提高了 4.2 °C。此外，通过建模研究获得了其嵌入双链体的迹象，并且在双链体形成时荧光发射强度显着降低。相比之下，单体Z没有明显的嵌入趋势得到证实在 DNA/RNA 杂合双链体中，如通过荧光的适度猝灭和相对于相应对照双链体的类似双链体热稳定性所表明的那样。用两个 15-mer 模型目标（DNA 和 RNA）研究了咔唑修饰的寡核苷酸对单核苷酸错配的识别效率。对于这两个系统，位于咔唑单体并列的错配显示热变性温度显着下降。加入Z单体的所有错配双链体的稳态荧光发射研究通常显示出有效的荧光猝灭。