| Secondary structure |
SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) |
Method for quantitative detection of local nucleotide flexibility. 2’-OH in flexible, unpaired nucleotides reacts preferentially with a probing reagent, forming adducts that can be identified as stops to primer extension by reverse transcriptase. |
| Secondary structure |
DMS (dimethylsulfate footprinting) |
DMS reacts with adenine at N1 and cytosine at N3. Reactive cytosines and adenines can be detected by reverse transcription and are considered as unpaired. |
| Secondary structure |
CMCT (1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate) |
CMCT reacts with N3 of uridine and, to a lesser extent, N1 of guanine. Reactive residues can be detected by reverse transcription and are considered as unpaired. |
| Secondary structure |
Kethoxal |
Kethoxal specifically attacks accessible N1 and N2 of guanine, and it is used for detection of unpaired guanines. The modified sites can be detected by reverse transcription. |
| Secondary structure + tertiary contacts |
Mutate-and-map |
SHAPE/DMS/CMCT chemical probing for a large number (preferably all) of point mutants of the RNA sequence. Analysis of changes in secondary structures of the set of point mutants can be used to infer tertiary contacts. |
| Solvent accessibility |
HRP (hydroxyl radical probing) |
Reports approximate backbone solvent accessibility. Solvent exposed nucleotides have high HRP reactivity. |
| Tertiary contacts |
MOHCA (multiplexed hydroxyl radical cleavage analysis) |
Enables the detection of pairs of contacting residues via random incorporation of radical cleavage agents. Contacting residues are detected from a cleavage pattern analyzed in two-dimensional gel electrophoresis. |
| Tertiary contacts |
Cross-linking |
Based on the formation of covalent bonds between spatially close regions of RNA that may be distant in sequence. Can be achieved using physical factors such as UV light or by chemical reagents. |
| Distances between labeled residues |
FRET (Förster Resonance Energy Transfer) |
Distances between fluorescent dyes linked to RNA molecule are inferred from the intensity of energy transfer. |
| Distances between labeled residues |
ESR/EPR (Electron Spin/Paramagnetic Resonance) spectroscopy |
Distances are derived from the measured spin–spin splittings for unpaired electrons localized on paramagnetic labels linked to RNA molecule |
| Global shape |
SAXS/SANS (Small Angle X-ray/Neutron Scattering) |
Provides information about the pair distance distribution within the molecule under study, which can be used to infer the particle envelope/shape. |
| Global shape |
Cryo-EM (Cryogenic Electron Microscopy) |
A 3D model is reconstructed through analysis of a very large number of 2D EM images |
