DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody. DNA G-Quadruplex Recognition In Vitro and in Live Cells by a Structure-Specific Nanobody

G-quadruplexes (G4s) are alternative DNA secondary structures of the human genome with key roles in transcription, replication and genome stability. Structure-specific small molecules and antibodies, such as BG4, have facilitated the detection of G4s in cells and chromatin. The availability of an alternative probe to BG4 would help to address some of its potential limitations in mapping G4 structures and would independently confirm and/or extend our earlier findings. Herein, we describe the development of SG4, a camelid single-chain only derived nanobody raised against the human MycG4 promoter structure. SG4 is small, easily expressed and demonstrates low nanomolar affinity for folded G4 structures in vitro. Clues to SG4 binding were obtained experimentally by mutating single amino acids. Moreover, AlphaFold2 protein predictions combined with molecular dynamics/docking simulations generated a de novo binding model of SG4-G4 interactions that identifies key amino acids with significant contributions to G4 binding. Using SG4 in G4 ChIP-seq, we also demonstrate the detection of G4 structures in two cancer cell lines. SG4 thus provides an alternative tool for G4 genome mapping and independently verifies our earlier findings. Overall design: Two cancer cells lines, U2OS and K562, have been profiled using a nanobody able to bind to DNA G4 secondary structures. For each cell line 3 biological replicates were performed and for each biological replicates 3 technical were prepared along with an input control.

G-四链体（G-quadruplexes, G4s）是人类基因组中的非典型DNA二级结构，在转录、复制及基因组稳定性调控中发挥关键作用。结构特异性小分子与BG4等抗体，已为细胞及染色质内G4s的检测提供了有力工具。开发BG4的替代探针，将有助于克服其在G4结构图谱绘制中存在的部分潜在局限，并可独立验证乃至拓展我们此前的研究成果。

本文报道了SG4的研发历程：SG4是一种骆驼源单链纳米抗体，以人类MycG4启动子结构为免疫原制备所得。该纳米抗体分子量小巧、易于重组表达，且在体外对折叠态G4结构展现出低纳摩尔级的结合亲和力。我们通过单点氨基酸突变实验，获取了SG4结合特性的相关实验线索。此外，结合AlphaFold2蛋白质结构预测与分子动力学/对接模拟，我们构建了SG4-G4相互作用的全新结合模型，该模型明确了对G4结合具有显著贡献的关键氨基酸残基。将SG4应用于G4染色质免疫沉淀测序（G4 ChIP-seq）实验后，我们还证实其可在两种癌细胞系中有效检测到G4结构。因此，SG4为G4基因组图谱绘制提供了新型替代工具，并独立验证了我们此前的研究成果。

整体实验设计：本研究针对U2OS与K562两种癌细胞系，采用可结合DNA G4二级结构的纳米抗体进行了图谱分析。每种细胞系均设置3次生物学重复，每一次生物学重复配套3次技术重复，并同步设置输入对照样本。