Due to the mounting evidence that RNA structure plays a critical role in regulating almost any essential physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 performs comparably well to NAI on naked RNA under in vitro conditions and it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo. Overall design: SHAPE-MaP Definitions: TGIRT-III/HIV RT/SSII: reverse transcriptase enzymes I5: Indoline-5-carboxylic acid imidazolide 1M4: 1-Methylimidazole-4-carboxylic acid imidazolide 6A3: 6-Aminopyridine-3-carboxylic acid imidazolide B5: Benzotriazole-5-carboxylic acid imidazolide NIC: Nicotinic acid imidazolide NAI: 2-Methylpyridine-3-carboxylic acid imidazolide 2A3: 2-Aminopyridine-3-carboxylic acid imidazolide