Table 1.

Summary of methods used to probe RNA secondary structure.

	Method	Biases/Limitations	RNA specificity	Mechanism of Method	Experimental Context	Refs
Adducts	DMS	A,C specific	ssRNA	Alkalates the N-1 in A and the N- 3 in C	in vivo and in vitro	2, 3, 55, 64, 96, 99, 135, 136, 185, 194
	Diethyl Pyrocarbonate	A specific	ssRNA	Carboxylates N-7 in A	in vitro	135, 136
	Hydrazine	U specific	ssRNA	Nucleophilic attack of U, removing the base	in vitro	135, 136
	NAI/NAI-N3	No bias, labels ribose sugar	ssRNA	Acylates 2’ hydroxyl of unpaired nucleotides	in vivo and in vitro	55, 119, 187
Nucleases	RNase A	Cleaves after purines	ssRNA	Leaves 5’OH and 3’P	in vitro	110, 173
	RNase T1	Preferrential cleavage after guanisines	ssRNA	Leaves 5’OH and 3’P	in vitro	110
	RNase U2	Cleaves after pyrimidines	ssRNA	Leaves 5’OH and 3’P	in vitro	81, 179
	RNase V1	None	dsRNA	Leaves 5’P and 3’OH	in vitro	38, 108
	Nuclease P1	None	ssRNA	Leaves 5’P and 3’OH	in vitro	27, 84
	Nuclease S1	None	ssRNA	Leaves 5’P and 3’OH	in vitro	27, 84
	RNase I	None	ssRNA	Leaves 5’OH and 3’P	in vitro	27, 84
Other	NMR	None	N/A	Aligns molecules in a magnetic field	in vitro	9, 12, 63, 195
	X-Ray Crystallography	Must be crystallizable RNA, in vitro folding only	N/A	Scatters X-rays in an interpretable pattern around an RNA crystal	in vitro	62, 79, 80, 147
	In silico algorithms	Difficult to predict in vivo folding	N/A	mostly predicts based on free energy and conservation	in silico	50, 51, 115, 197

Three categories of methods are covered; chemical adducts (red), RNases (green), and other (blue).