The instability of DNA triplexes particularly at neutral pH and above severely limits their applications. Here, we demonstrate that the introduction of a thiazole orange (TO) intercalator onto a thymine nucleobase in triplex forming oligonucleotides (TFOs) resolves this problem. The stabilising effects are additive; multiple TO units produce nanomolar duplex binding and triplex stability can surpass that of the underlying duplex. In one example, a TFO containing three TO units increased the triplex melting temperature at pH 7 by a remarkable 50 °C relative to the unmodified triplex. Notably, TO intercalation promotes TFO binding to target sequences other than pure polypurine tracts by the use of 5-(1-propynyl)cytosine (pC) against C:G inversions. By overcoming the instability of triplexes across a broad range of pH and sequence contexts, these very simple ‘TOTFO’ probes could expand triplex applications into many areas including diagnostics and cell imaging.

中文翻译：

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荧光噻唑橙 TOTFO 探针可在 pH 7 及以上稳定平行 DNA 三链体†

DNA 三链体的不稳定性，特别是在中性 pH 值及以上的情况下，严重限制了它们的应用。在这里，我们证明，在三链体形成寡核苷酸（TFO）中的胸腺嘧啶核碱基上引入噻唑橙（TO）嵌入剂可以解决这个问题。稳定效果是相加的；多个 TO 单元产生纳摩尔双链体结合，三链体稳定性可以超过基础双链体。在一个例子中，相对于未修饰的三链体，含有三个 TO 单元的 TFO 将 pH 7 时的三链体熔化温度显着提高了 50 °C。值得注意的是，TO 嵌入通过使用 5-(1-丙炔基)胞嘧啶 (pC) 对抗 C:G 倒置，促进 TFO 与纯多嘌呤束以外的靶序列结合。通过克服三链体在广泛的 pH 和序列环境中的不稳定性，这些非常简单的“TOTFO”探针可以将三链体应用扩展到包括诊断和细胞成像在内的许多领域。