Table 3.
Experimental methods for uncovering the interaction between proteins and RNAs.
| Classes | Methods | Advantages | Disadvantages | References |
|---|---|---|---|---|
| In the test tube | EMSA | Separation of numerous types of complexes, such as monomer and dimer; Works well with crude cell extracts. | Low-throughput; Failure of detecting binding sites. | (125, 127) |
| RNA pull-down | A simple protocol; Enrichment of low-abundance RBPs. | Failure of confirming direct or indirect interaction; Failure of forming RNA-protein interactions that only occur in vitro under non-physiological conditions. | (125, 128) | |
| SELEX | Direct interaction of the oligonucleotides with the target is closely application-oriented; Independence of in-depth knowledge regarding the respective target for aptamer selection. | No standardized aptamer selection protocol for target. | (129–132) | |
| RNAcompete | Accurate estimation of the relative preference for a large numbers of individual sequences; Querying preferences are available for structured RNA; Time-saving. | Limited size of the current RNA pool for the represented combinations of sequence and structure. | (125, 133) | |
| RBNS | Accurate estimation of dissociation constants of RBP-RNA complex; A more reliable prediction of RNA folding and a better identification of the binding structural determinants than RNAcompete. | N/A | (125, 134, 135) | |
| In cells | RIP Assay | A simple and standard protocol; Preserves the intracellular native complexes. | Failure of confirmation of direct or indirect interaction between RNA and protein; Failure of determination of the precise site within the RNA interacting protein; Poor resolution; Antibody-consuming. | (125, 136) |
| CLIP | A sensitive determination of binding sites. | Low abundance of RNA-ribonucleoprotein complexes; Potentially inefficient library preparation; Large amounts of raw material required. | (108, 125, 137) | |
| PIP-seq | A simultaneous view of the global landscapes of both RNA secondary structure and RNA-protein interactions. | High concentrations of structure-specific RNases. | (138) |
EMSA, Electrophoretic Mobility Shift Assay; SELEX, Systematic Evolution of Ligands by Exponential Enrichment; RBNS; RNA Bind-n-Seq; RIP, RNA Immunoprecipitation; CLIP, Cross-linked Immunoprecipitation; PIP-seq, Protein Interaction Profile Sequencing; N/A, not applicable.