Table 2.

Overview of techniques to target a specific RNA of interest to study its interacting proteins.

Method	Advantage	Disadvantage	Cell Number
Antisense Probe-Based Methods
PAIR [74]	Direct interactors RBPs of a specific region Different transcript isoforms Denaturing purification conditions	Difficult to study whole interactome of a specific RNA Potential co-purification of naturally biotinylated proteins Costs of CPP-PNA-Bpa probes	2 × 106–1 × 107 cells
CHART-MS [75]	Different transcript isoforms Denaturing purification conditions	Direct and indirect interactors RNase H assay to determine accessibility of the probes Potential co-purification of naturally biotinylated proteins	8 × 107 cells
ChiRP-MS [10]	No prior knowledge of the RNA structure and accessibility required Capture of RNA with impaired integrity Denaturing purification conditions	Direct and indirect interactors Large number of probes may result in higher background contamination Potential co-purification of naturally biotinylated proteins Costs of large number of probes	1–5 × 108 cells depending on the cell type
RAP-MS [11]	Direct interactors No prior knowledge of the RNA structure and accessibility required Long probes may result in high specificity Capture of RNA with impaired integrity Denaturing purification conditions	Large number of probes may result in higher background contamination Potential co-purification of naturally biotinylated proteins Costs of large number of probes Cost of synthesizing long probes	2 × 107 cells
LNA/DNA mixmers [76]	Direct interactors Different transcript isoforms Increased hybridization specificity Denaturing purification conditions	Cost of LNA-containing probes Use of only one probe results in RNA integrity to be critical 20% recovery Less optimal for low-copy-number transcripts	2000 cm2 of HeLa cells at 70% confluence
HyPR-MS [77]	Cost- and labor-effective protocol Reduced sample and background variance Different transcript isoforms Denaturing purification conditions	Direct and indirect interactors Potential co-purification of naturally biotinylated proteins	1 × 108 cells
PPR proteins [78]	Different transcript isoforms	Direct and indirect interactors Efficacy only proven in chloroplasts and mitochondria Restriction to use denaturing purification conditions	Chloroplasts isolated from 40 g tissue
Biotin-tagged Cas9 purification [79]	Different transcript isoforms	Direct and indirect interactors Restriction to use denaturing purification conditions Potential co-purification of naturally biotinylated proteins	5x106 cells