Long non-coding RNAs (lncRNAs) are a type of RNA molecule that are longer than 200 nucleotides and do not encode proteins. Despite their lack of protein-coding capacity, lncRNAs play critical roles in various biological processes, including transcriptional regulation, chromatin modification, and cellular differentiation. However, compared to protein-coding genes, the sequence conservation of lncRNAs is relatively low, making it challenging to predict their functional roles.
Moreover, predicting the spatial folding structure of lncRNAs is not straightforward, as they tend to adopt complex tertiary structures. In addition, the interaction between RNA and DNA is not solely based on the Watson-Crick base pairing rules that govern the binding of complementary nucleotides, making it challenging to predict the binding sites of lncRNAs in the genome. Therefore, developing experimental methods to capture lncRNAs and their binding partners in the nucleus is essential for understanding their functions and mechanisms of action.
One such experimental method is the RNA Antisense Purification (RAP) technique. RAP uses long probes of 120 nucleotides to capture lncRNAs and their binding partners in vivo. The long probes provide the advantage of reducing background noise and enabling the capture of lncRNAs that may have complex secondary or tertiary structures.
Moreover, RAP does not require prior knowledge of the binding domains involved in chromatin interactions with lncRNAs. Instead, oligonucleotides are tiled across the entire target RNA, ensuring that all potential hybridization sites are fully utilized. This feature allows RAP to capture lncRNAs and their binding partners even in the context of extensive protein-RNA interactions, RNA secondary structure, or partial RNA degradation.
Creative Proteomics provides RAP-Seq technology for capturing lncRNAs and their binding partners in the nucleus, thereby enabling the study of their functional roles and mechanisms of action.
RAP is a method that separates lncRNA and maps its target DNA sequence through a probe capture mechanism. First, cells are cross-linked and lysed before chromatin digestion into 100-300 bp DNA fragments using DNase I. A biotinylated RNA probe, antisense to the lncRNA, hybridizes with streptavidin and captures the target RNA. The biotin-RNA probe is 120 nt long and is tiled every 15 nt along the span of the lncRNA. The captured complex is washed and prepared for sequencing. RNA library preparation is performed using RAP-RNA, while DNA library preparation is performed using standard chromatin immunoprecipitation (ChIP).
Probe design: design antisense DNA splicing probes for selective extraction of RNA targets by RAP
Cell collection: harvesting cells for RAP experiments
Cell cross-linking: cross-linking of cells and collection of cell pellets
Cell lysis: Lysis of cross-linked cells to prepare cell lysate
Ultrasonication: shear DNA by sonicating cross-linked cell lysates
RAP: hybridize biotinylated DNA probes to RNA and isolate bound chromatin
RNA isolation: Extraction of RNA fraction from RAP samples, quantification by RT-qPCR to determine enrichment efficiency, and identification by sequencing or qPCR
DNA isolation: DNA fragments are extracted from RAP samples and identified by sequencing or quantitative PCR
Overview of one-to-all methods to capture RNA-interacting genomic loci (Kato et al., 2020).
Creative Proteomics has accumulated a wealth of experience in RNA-protein interaction research. Our team of technical professionals also offers other techniques for RNA-protein interaction analysis, including ChIRP, CHART, RIP and RNA Pull Down, etc. Contact us to learn more.