含修饰碱基 Z 的平行三链形成寡核苷酸可序列特异靶向富 GC 基因位点
Sequence-specific targeting of GC-rich gene loci by parallel triplex-forming oligonucleotides containing a modified nucleobase
研究团队用含合成碱基 6-amino-5-nitropyridin-2-one（Z）的平行结合型三链形成寡核苷酸（TFO），实现了对富 GC 基因位点的序列特异靶向。Z 使 TFO 能在不依赖 pH 的条件下识别 G-C 碱基对，从而突破天然碱基在细胞环境中识别能力有限的限制。作者以 MYC 启动子内两个结构不同的调控元件为靶点，在游离报告基因实验和 endogenous 基因位点上均观察到启动子活性被抑制 50%–80%，其中抑制最强的是可形成四链、作为转录因子招募结构枢纽的富 GC 元件。
来源：Nucleic Acids Research · doi.org
Targeting GC-rich gene loci is a major challenge owing to their high duplex stability, repetitive sequence composition, and propensity to adopt alternative DNA structures. Triplex-forming oligonucleotides (TFOs) provide a programmable strategy toward the recognition of GC-rich DNA, but their application is restricted by the limited recognition capabilities of natural nucleobases in a cellular setting. Here, we overcome this barrier using parallel-binding TFOs containing the synthetic nucleobase 6-amino-5-nitropyridin-2-one (Z), which enables pH-independent recognition of G-C base pairs. Using two structurally distinct regulatory elements within the MYC promoter, we show that Z-modified TFOs form stable, sequence-selective triplexes that repress promoter activity by 50%–80% in both episomal reporter assays and at endogenous gene loci. Notably, the greatest repression was observed at a GC-rich quadruplex-forming element that functions as a structural hub for transcription factor recruitment. To our knowledge, this represents the first demonstration that a simple nucleobase modification alone is sufficient to enable parallel-binding TFOs to repress expression of an endogenous gene, establishing a general strategy for targeting GC-rich regulatory elements through programmable DNA recognition.