iMbody - a nanobody for i-Motif research. iMbody - a nanobody for i-Motif research

i-Motifs (iMs) are four-stranded structures formed by certain cytosine-rich nucleic acids. They may be involved in regulation of cellular processes such as transcription. In addition, the pH dependency of iMs makes them suitable for certain nanobiotechnological applications. Probes that recognize these structures with high specificity and affinity can significantly advance the iM research field. Here we report the generation of an iM specific nanobody (iMbody) and describe its applications for the in vitro characterization of a potential iM-forming oligonucleotide, strand-specific pull-down of iMs present in the human genome at near physiological pH and temperature followed by sequencing, and visualization of iMs in the nuclei of human cells. Utilization of iMbody could be beyond above-mentioned applications and it can be used to investigate iMs in other organisms. The plasmid encoding iMbody for the bacterial expression is available via Addgene (Plasmid #184496). Overall design: i-Motif specific nanobody (iMbody) was used to perform strand-specific pull-down of i-Motifs present in the genomic DNA purified from K562 cells followed by high-throughput sequencing. Pull-down was performed at pH 7.2 ± 0.1 and 37 °C. Two replicates included in the study.

i-基序（i-Motifs，简称iMs）是一类由特定富含胞嘧啶的核酸形成的四链结构。该结构可能参与转录等细胞过程的调控。此外，iMs的pH依赖性使其可应用于部分纳米生物技术场景。能够高特异性、高亲和力识别此类结构的探针，可显著推动iM研究领域的进展。本研究报道了一种iM特异性纳米抗体（iMbody）的构建，并详述其多项应用：包括对潜在可形成iM的寡核苷酸进行体外表征、在接近生理pH与温度的条件下对人类基因组中存在的iM开展链特异性亲和下拉并辅以高通量测序，以及在人类细胞核内实现iM的可视化。iMbody的应用潜力不限于上述场景，还可用于探究其他生物体内的iM。用于细菌表达的iMbody编码质粒可通过Addgene获取（质粒编号#184496）。整体实验设计：使用iM特异性纳米抗体（iMbody）对从K562细胞中提取的基因组DNA所含的i-基序进行链特异性亲和下拉，随后进行高通量测序。该亲和下拉实验在pH 7.2 ± 0.1及37℃条件下开展，本研究共设置两个生物学重复。