RNA Guanine-quadruplexes (rG4s) are important nucleic acid structures that govern vital biological processes. Although numerous tools have been developed to target rG4s, few specific tools are capable of discerning individual rG4 of interest. Herein, we design and synthesize the first L-aptamer–antisense oligonucleotide (ASO) conjugate, L-Apt.4–1c-ASO15nt(APP), with a focus on recognizing the amyloid precursor protein (APP) rG4 region as an example. The L-aptamer module binds with the rG4 structure, whereas ASO hybridizes with flanking sequences. Together, these two modules enhance the precise recognition of APP rG4. We demonstrate that the L-Apt.4–1c-ASO15nt(APP) conjugate can interact with the APP rG4 region with sub-nanomolar binding affinity, and distinguish APP rG4 from other G4s and non-G4s in vitro and in cells. We also show that L-Apt.4–1c-ASO15nt(APP) can inhibit APP protein expression. Notably, we investigate the inhibitory mechanism of this newly developed tool, and reveal that it controls gene expression by hindering DHX36 protein from unraveling the rG4, as well as by promoting translational inhibition and RNase H-mediated mRNA knockdown activity. Our novel L-aptamer–ASO conjugate tool not only enables the specific recognition of rG4 region of interest, but also allows efficient gene control via targeting rG4-containing transcripts in cells.

中文翻译：

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通过 L-适配体-D-寡核苷酸偶联物在体外和细胞中选择性识别 RNA G-四链体


RNA 鸟嘌呤-四链体 （rG4） 是控制重要生物过程的重要核酸结构。尽管已经开发了许多靶向 rG4 的工具，但很少有特定工具能够识别单个感兴趣的 rG4。在此，我们设计并合成了第一个 L-适配体-反义寡核苷酸 （ASO） 偶联物 L-Apt.4-1c-ASO15nt（APP），重点是识别淀粉样蛋白前体蛋白 （APP） rG4 区域作为示例。L-适配体模块与 rG4 结构结合，而 ASO 与侧翼序列杂交。这两个模块共同增强了对 APP rG4 的精确识别。我们证明 L-Apt.4-1c-ASO15nt（APP） 偶联物可以以亚纳摩尔结合亲和力与 APP rG4 区域相互作用，并在体外和细胞中区分 APP rG4 与其他 G4 和非 G4。我们还表明 L-Apt.4–1c-ASO15nt（APP） 可以抑制 APP 蛋白表达。值得注意的是，我们研究了这种新开发的工具的抑制机制，并揭示了它通过阻止 DHX36 蛋白解开 rG4 以及促进翻译抑制和 RNase H 介导的 mRNA 敲低活性来控制基因表达。我们新颖的 L-适配体-ASO 偶联工具不仅可以特异性识别感兴趣的 rG4 区域，还可以通过靶向细胞中含有 rG4 的转录本来实现高效的基因控制。