Table 2.

Common Biophysical Techniques That Have Been Used for lncRNA, Longer RNA Transcripts or Could Be Applied in Future Studies^a

technique	shorthand	values solved	pros	cons	RNA examples	ref
electrophoretic mobility shift assay	EMSA	Kd	low-cost equipment	often requires P-32 radiolabel	HOTAIRb; Xistb	71
filter-binding assay		Kd	straight-forward method; efficient	requires radiolabeling (P-32); nonspecific binding to filter; aggregation of materials not related to native binding event	HOTAIRb	89
surface plasmon resonance	SPR	Kd	label-free	target immobilization; cannot report on 2+ state binding model	PARTICLEb	90
isothermal titration calorimetry	ITC	n, Kd, ΔH, ΔG, ΔS	label-free; kinetic and thermodynamic information; can report on 2+ state systems	high concentrations of materials	HIV-1 TARc; purine riboswitchc; 16S rRNAc	91–93
Förster resonance energy transfer	FRET	n, Kd	relatively cheap; can measure distance relationship; sensitive	requires FRET pairs that do not interfere with RNA structure; depends on conformational change and may miss static binding events.	rRNAc, tRNAc	94
fluorescence polarization/anisotropy	FP/FA	n, Kd, aggregation, folding dynamics	no energy transfer requirement; can be HTP	requires fluorescent labeling; binding must significantly change the size of the fluorophore-labeled molecule	Tetrahymena Group I Intron Ribozymec	95, 96

^aHTP = high-throughput; n = Hill’s coefficient; K_d = dissociation constant; ΔH = change in enthalpy; ΔS = change in entropy; ΔG = change in Gibb’s free energy.

^bLong ncRNA example.

^cOther ncRNA example.