Table 1.
Methods to study RNA structure and RNA-protein interactions.
| NMR |
| Nuclear magnetic resonance spectroscopy can be a very powerful tool for studying RNA structures in fine detail with a high confidence. It is advantageous to X-Ray crystallography techniques as RNA molecules can be studied in a more natural state while dissolved in solution, however it relies on large preparations of highly pure and uniform RNA and is generally restricted to solving small discrete structures. |
| PARS |
| This technique brings together classic RNA footprinting techniques with next-generation sequencing. It involves treating RNA independently RNase V1 and S1 nucleases which cut double and single stranded RNA respectively. Cleaved fragments are adaptor ligated and sequenced allowing a map to be generated of single and double stranded RNA down to single nucleotide resolution. |
| DMS |
| DMS treatment modifies RNA by adding a methyl group to any unpaired or loosely structured A and C bases in a sample. Once methylated the bases can no longer form base pairs and will cause cDNA transcripts to terminate early. When compared to a non-DMS treated sample the sites of early termination, and thus the presence of unpaired bases can be deduced. The addition of next generation sequencing (DMS-seq/Structure-seq) greatly increases the power of the technique and allows the rates of base modification to be mapped in a quantitative manner. Targeted Structure-seq improves the specificity and power of the technique by using primers targeted for the length of a specific RNA of interest instead of sequencing the whole transcriptome. |
| SHAPE |
| SHAPE methods use chemical reagents which selectively modify flexible or unpaired bases by forming adducts on the 2'-hydroxyl of the RNA backbone. As with other modifications these adducts will result in the early termination reverse transcription. As the reagents only modify the RNA backbone, they have the advantage of being independent of base identity and provide a reliable measurement of individual nucleotide flexability. SHAPE-Map uses specialized conditions for reverse transcription which result in the misreading of SHAPE-modified nucleotides and the introduction of non-complementary base mutations instead of early termination. These mutations are easily identified after sequencing and their relative frequencies can be mapped to the reference sequence. |
| PARIS |
| PARIS works by fixing the base pairs of dsRNA of cells in vivo using the specific and reversible nucleic acid cross-linker called AMT. After cross-linking, samples undergo proteinase treatment and partial RNA degradation. Subsequently they are gel purified from a 2D gel, leaving only fixed dsRNAs. dsRNA then undergoes proximity ligation and undergo next generation sequencing. The resulting reads represent all the native dsRNA in the organism and can be mapped to infer their structure. |
| RIP |
| RNA immunoprecipitation takes advantage of antibodies to pull down RNA bound to a given protein. The technique cannot differentiate between direct and indirectly bound RNA and may also generate false positives from interactions that occur after cell lysis. |
| CLIP |
| Improves the specificity of RIP by UV crosslinking of RNA/protein complexes before extraction. This allows the removal of weakly bound RNA through stringent washing. The remaining RNA can then undergo reverse transcription and PCR amplification (or next generation sequencing). The main drawback of this method is the loss of a significant proportion of transcripts which are stalled at the cross-linking site resulting in truncated cDNAs. UV crosslinking can also introduce some bias as its ability to bind RNA to protein varies depending on the base/aa mediating the interaction. |
| iCLIP |
| Individual-nucleotide-resolution CLIP (iCLIP) was developed to enable recovery of truncated cDNAs lost in conventional CLIP. This is achieved by the circularization of cDNA after reverse transcription, attaching a new barcoded adaptor to the truncated end allowing it to be amplified after linearization. Barcode filtering allows truncated cDNAs to be identified along with the crosslinked nucleotide. Subsequent mapping of these small fragments to the reference sequence can be difficult. |
| eCLIP |
| Enhanced CLIP improves library preparation and circular ligation steps of iCLIP allowing greater power in filtering and mapping truncated sequences. |