Table 3.

Comparison of protein-nucleic acid approaches.

RNA	RNA centric – (identifying the proteins interacting with an RNA of interest)
	Method	Brief Description	Pros	Cons	Ref
	RAP-MS, PAIR, TRIP, CHART, ChIRP	UV or formaldehyde cross-linking followed by RNA pulldown using biotinylated nucleic acid probes.	No genetic engineering or exogenous expression of components. UV crosslinking is highly specific for direct RNA-protein interactions.	Cross-linking, UV in particular, has low efficiency, requiring 108-109 cells. FA is less specific and results in protein-protein crosslinks that increase background. DNA probes must be optimized and can lead to nonspecific capture of RNAs or contribute to lower efficiency.	119-123
	MS2-Biotrap	UV cross-linking followed by RNA pulldown via MS2-coat protein interaction.	Improves the pulldown workflow by avoiding ASO capture.	Cross-linking is low efficiency, requiring 108-109 cells. Exogenous expression of MS2-tagged RNA may not recapitulate physiological concentrations or conditions	124
	RaPID	MS2-modified endogenous RNA recruits a coat protein-PL enzyme fusion to biotinylate proteins interacting with the RNA of interest.	Avoids crosslinking and associated problems. Enables direct biotinylation of interacting proteins. Can be applied in vivo.	Biotinylation of the general location necessitates spatial references (e.g. scrambled RNA control) to eliminate false positives. PL captures indirect interactors. Exogenous expression of MS2-target RNA may not recapitulate physiological concentrations or conditions. Biotinylated proteins may be proximal to the MS2 site and not the RNA in general, making this method better for shorter RNAs.	24,125,126
	CRUIS, CBRPP, CARPID, dCas13d-dsRBD-APEX2	Proximity labeling enzyme (PafA / BioID/ BASU/ APEX2) fusion to catalytically inactive dCas13 to biotinylate proteins interacting with an endogenous transcript.	Enables direct biotinylation of proteins interacting with endogenous RNA transcripts. In vivo compatible and can be easily engineered for different targets. Avoids crosslinking.	Incomplete localization of Cas 13 can produce high background. May require guide optimization, as well as spatial references (non-targeting guide) to account for non-specific labeling. PL captures indirect interactors. Biotinylated proteins are proximal to the guide RNA site, and not the entire target RNA in general.	126,129
	Protein centric - (identifying the RNAs interacting with a protein of interest)
	Method	Brief Description	Pros	Cons	Ref
	CLIP-Seq, eCLIP, iCLIP, irCLIP PAR-CLIP, fCLIP	Cross-linking Immuno-Precipitation. There are many variations of the CLIP-seq protocol, but generally, crosslinking of proteins to RNA is carried out by UV (CLIP-seq), by UV using incorporated thiouridine (PAR-CLIP), or using FA (fCLIP). A protein of interest is isolated by antibody pulldown, and the covalently bound RNA is sequenced.	UV crosslinking is highly specific. Does not require genetic engineering or exogenous expression of components.	Can be difficult to obtain enough cross-linked RNA due to low efficiency of crosslinking, poor antibody pull-down, or low abundance of the RNA-RBP complex. Requires IP-grade antibodies.	106-111
	RIP-seq	Antibody pulldown of a protein of interest under non-denaturing conditions to recover the associated RNAs.	Higher RNA yield than CLIP. Simple protocol without genetic engineering or exogenous expression.	Lower signal to noise than CLIP, may capture indirect interactors, and has a higher chance of false positives due to FA crosslinking.	148
	RNA Tagging, TRIBE	RNA Tagging uses a poly-U-polymerase fused with the POI to extend poly uracil at the 3’ end of proximal RNAs, which can be subsequently enriched using poly-A ASO capture. TRIBE uses ADAR fused with the POI and mediates A to I editing of interacting RNAs, which can then be identified by sequencing.	Does not require antibody purification. Does not require crosslinking.	Exogenous expression of RBPs can lead to false positives/negatives. RNA tagging may be biased towards 3’ interactors.	149,150
	APEX-RIP, Proximity - Clip	Proteins are biotinylated by APEX2 labeling, and RNA and Proteins are crosslinked by UV and 4SU (proximity CLIP) or FA (APEX-RIP). Streptavidin pulldown enables the enrichment of RNA of a specific subcellular location.	Does not rely on antibody purification. Can recover organelle or location- specific RNAs. UV crosslinking captures direct interactors.	Formaldehyde crosslinking results in poor specificity, which can be overcome by UV crosslinking at the expense of efficiency. Adapting this method to RBP-specific capture necessitates IP-grade antibodies or genetic-tagging of RBP of interest.	113,114
	APEX-seq, CAP-seq	Proximity labeling of RNAs directly by a proximity labeling enzyme enables the enrichment of RNA that interacts with a POI or located in specific subcellular locations.	Direct labeling of RNA improves workflow, specificity, and efficiency. Can be performed in vivo.	Proximity labeling can capture indirect interactors. Adapting these techniques to studying specific RBPs requires exogenous expression of the RBP-PL fusion protein.	115-118
DNA	Protein centric – (identifying the DNAs associated with a protein of interest)
	Method	Brief Description	Pros	Cons	Ref
	ChIP-Seq	Chromatin Immuno-Precipitation. Antibody pulldown of a POI under non-denaturing conditions allows the identification of associated DNA fragments.	Widely adopted and straight-forward protocol, relatively unbiased, and does not require exogenous expression.	Requires IP-grade antibodies.	131
	ALaP	APEX2 is fused to a protein of interest to detect associated DNA.	Does not require antibody pulldown.	Requires a spatial reference to improve SNR. Exogenous expression of fusion protein may not reflect physiological conditions.	132
	Chromatin modification centric – (identifying proteins associated with a specific chromatin modification)
	ChromID	Fusion of BASU promiscuous biotin ligase to ‘reader domains’ that specifically bind to chromatin modifications (e.g. H3K4me3), which enables the identification of proteins associated with specific chromatin modifications.	Direct labeling of proteins associated with a specific chromatin modification.	Overexpression of the reader domains may perturb the normal occupancy of chromatin modifications. Proximity labeling may require a spatial reference to improve SNR.	139
	DNA centric – (identifying proteins associated with a specific DNA sequence)
	RIME, ChIP-MS	DNA-protein crosslinking followed by immunoprecipitation.	Enable the assessment of chromatin-bound protein complexes.	Crosslinking has low efficiency and may result in false positives.	137,138
	(APEX-DNA Binding Protein fusion)	Fusion of a PL enzyme to a DNA binding protein (DBP) enables the labeling of proteins associated with the DNA-binding site of the DBP.	Does not require crosslinking or antibody pulldown. Can be performed in vivo.	May require a spatial reference to improve SNR. Exogenous expression of a DNA binding protein can perturb the studied system.	39
	CASPEX, C-BERST	APEX2-dCas9 fusion proteins are expressed in a cell along with targeting guides to enable labeling of proteins associated with a specific DNA sequence.	Easily reprogrammed and simple protocol. Proteins can be directly enriched and avoids crosslinking or IP.	May requires a spatial reference (e.g. non-targeting guide) to improve SNR. Exogenous expression of Cas9 can perturb the studied system.	134,135

Abbreviations: PL – proximity labeling; FA – formaldehyde; RBP – RNA binding protein; SNR – signal to noise ratio; POI – protein of interest.