Table 1.
Recent technologies applied to the microRNA (miRNA) pathway
| Methods | General attributes | Recent applications to the miRNA pathway |
|---|---|---|
| High-throughput substrate screening | • Similar to classical in vitro selection assays. • Often involves parallel processing of a large endogenous substrate pool and/or library of designed or randomized variants. • Deep sequencing is used as a readout, to infer functionally relevant features for processing. |
• pri-miRNA features30,31,36 • pre-miRNA features38,54 |
| Cryo-electron microscopy (Cryo-EM) | • Uses an electron beam to image a specimen under cryogenic conditions. • Data are processed into EM densities that can be used to assign atomic coordinates. • Advantages include that Cryo-EM is suitable for proteins or complexes of large molecular weight; relatively small amounts of sample are needed; multiple conformational states can be captured in a single experiment; and no need for crystallization. |
• Mammalian Drosha/DGCR8 complex86,87 • Mammalian Dicer complexes81,90,91 • Drosophila Dicer complexes: Dicer-192 and Dicer-295,96 • Arabidopsis Dicer complexes: DCL193 and DCL397 |
| Single-molecule assays | • Offers real-time dynamics of biological reactions. • In vitro assays usually involve purified materials. • Observation times depend on the reaction kinetics, photostability, and lifetime of fluorophores, but can go down to microseconds. • Detection is generally diffraction limited (~200-300 nm spatial resolution). However, single-molecule Förster resonance energy transfer (smFRET) operates at 1-10 nm, thus resolving inter and intra-molecular motions. • Single-molecule imaging in living cells is typically done with fluorescently-tagged molecules and may utilize multimeric tags or scaffolds to enhance detection. |
• Dynamic interplay of human Dicer and TRBP94 • In vitro target search and interrogation by human Ago2/RISC complexes102–105,107 • Live cell imaging of targeting and regulation by human Ago2108–110 • Assembly and dynamics of Drosophila AGO2/RISC complexes100,101 |
| RNA bind-n-seq (RBNS) | • Yields relative quantitative binding affinities of an RNA binding protein (RBP) across a library of target sites. • Typically, a purified RBP is incubated with a randomized pool of RNAs. Co-purified RBP targets are then analyzed by deep sequencing to identify their features. |
Ago2–miRNA complex binding affinity to target RNAs64,65 |
| CRISPR–Cas9 screening | • Genetic strategies for mutagenesis can be applied to identify factors involved in a cellular process of interest. • miRNA screening often incorporates a specific reporter as a functional readout, enabling cell sorting before deep sequencing of enriched or depleted guide RNAs. |
• ERH/SAFB2 in miRNA cluster assistance153 • ZSWIM8 in target-directed miRNA degradation (TDMD)193,194 |