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.