Молекулалық биологиядағы РНҚ байланыс орындарын анықтау әдісі

Method used in molecular biology
Cross linking and immunoprecipitation (CLIP, or CLIP seq) is a method used in molecular biology that combines UV crosslinking with immunoprecipitation in order to identify RNA binding sites of proteins on a transcriptome wide scale, thereby increasing our understanding of post transcriptional regulatory networks. CLIP can be used either with antibodies against endogenous proteins, or with common peptide tags (including FLAG, V5, HA, and others) or affinity purification, which enables the possibility of profiling model organisms or RBPs otherwise lacking suitable antibodies.

CLIP begins with the in vivo cross linking of RNA protein complexes using ultraviolet light (UV). Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity (on the order of Angstroms apart). The cross linked cells are then lysed, RNA is fragmented, and the protein of interest is isolated via immunoprecipitation. In order to allow for priming of reverse transcription, RNA adapters are ligated to the 3' ends, and RNA fragments are labelled to enable the analysis of the RNA protein complexes after they have been separated from free RNA using gel electrophoresis and membrane transfer. Proteinase K digestion is then performed in order to remove protein from the crosslinked RNA, which leaves a few amino acids at the crosslink site. This often leads to truncation of cDNAs at the crosslinked nucleotide, which is exploited in variants such as iCLIP to increase the resolution of the method. cDNA is then synthesized via RT PCR followed by high throughput sequencing followed by mapping the reads back to the transcriptome and other computational analyses to study the interaction sites.

eCLIP (enhanced CrossLinking and ImmunoPrecipitation followed by high throughput sequencing) is also used to map RBP binding sites on RNAs transcriptome wide. eCLIP was designed to improve upon iCLIP by increasing the efficiency in converting purified RNA fragments into cDNA library. At its publication, eCLIP was reported to increase such efficiency by >1000 fold, which not only decreases wasted sequencing of PCR duplicate molecules, but also dramatically decreases experimental failures during the CLIP procedure. Additionally, the amplification in eCLIP is now comparable to RNA seq, enabling rigorous quantitative normalization against paired input controls (to remove background at ribosomal and other highly abundant RNAs) as well as quantitative comparison across peaks and samples, enabling the ability to detect allele specific binding or differential RNA binding between conditions. As in other CLIP methods, eCLIP relies on RBP RNA interactions covalently linked using UV crosslinking of live cells. Cells are then lysed, and RNA is fragmented using limited RNase treatment. A specific RBP (and its bound RNA) is then immunoprecipitated using an antibody that specifically recognizes the targeted RBP. After ligation of a 3’ RNA adapter, immunoprecipitated material (as well as a paired input sample) are run on denaturing protein gels and transferred to nitrocellulose membranes. A region from the protein size to 75 kDa above is cut from the membrane and treated with Proteinase K to release RNA. After cleanup, RNA is then reverse transcribed to ssDNA, when a second adapter is ligated. By ligating the second adapter to cDNAs, eCLIP can identify truncated cDNAs, similar to iCLIP, and thereby study RNA protein interaction sites with high resolution. PCR amplification is then used to obtain sufficient material for high throughput sequencing. eCLIP can also be used to identify miRNA targets and profile RNA modifications such as m6A. eCLIP datasets have been produced for over 150 RBPs with validated commercially available antibodies.

sCLIP (simple CLIP) is a technique that requires lower amounts of input RNA and omits radio labeling of the immunoprecipitated RNA. The method is based on linear amplification of the immunoprecipitated RNA and thereby improves the complexity of the sequencing library despite significantly reducing the amount of input material and omitting several purification steps. Additionally, it permits a radiolabel free visualization of immunoprecipitated RNA by using a highly sensitive biotin based labeling technique. Along with a bioinformatical platform this method is designed to provide deep insights into RNA–protein interactomes in biomedical science, where the amount of starting material is often limited (i. e. in case of precious clinical samples). Additional iCLIP variants have also been developed that retain the individual nucleotide resolution but differ in one or more steps from the original iCLIP method. These include iCLIP2, irCLIP, iiCLIP, and iCLIP1.5, a few to name. As a modification of CLIP, methylated RNA sites were identified with the use of mutant enzyme or modification specific antibody with the methods termed miCLIP or m6A CLIP. Certain steps are difficult to optimize and frequently have low efficiencies. For example, overdigestion with RNase can decrease the number of identified binding sites and thus needs to be optimised. Crosslinking efficiency also varies between proteins, and nucleotide bias of crosslinking has been reported, for example by comparing cross linking sites and motifs enriched when protein RNA complexes are studied in vivo in living cells and in vitro, though methods are being developed to minimise such bias for enriched motif discovery. Computationally predicted miRNA targets derived from TargetScan are comparable to CLIP in identifying miRNA targets, raising questions as to its utility relative to existing predictions. Because CLIP methods rely on immunoprecipitation, crosslinked RNA could in some cases affect antibody epitope interactions. Finally, significant differences have been observed. Therefore, raw CLIP results require further computational analyses to thoroughly investigate RNA protein binding site interactions within the cell.