SHAPE CATCHER: To determine the secondary structures of RNAs in their natural cellular context (1), an unpaired nucleotide (orange) is first modified with NAI-N3 (2). Next, biotin clicks onto the NAI-N3 (3) allowing retrieval of the modified RNA via biotin binding to a streptavidin bead. The RNA is then chopped into small fragments (4) for sequencing to reveal which nucleotide is modified and thus unpaired (orange) versus unmodified and thus paired or bound to a protein (green). If these in vivo modifications, analyzed across the transcriptome and among many copies of each RNA, are compared with those seen on the same RNAs in vitro (5), potential protein binding sites (1) and other structural differences can be revealed because the probe will bind to different bases.© GEORGE RETSECK
Like pieces of sticky tape, single-stranded RNA molecules can fold over and bind to themselves, via base pairing. Determining the complicated three-dimensional structures such folding creates can reveal the RNAs’ functions, but classic techniques for investigating molecular structure, such as X-ray crystallography, are “very laborious” in that they “can only be used on one RNA at a time,” says Howard Chang of Stanford University.
To study the secondary structures of multiple RNAs simultaneously, researchers have come up with chemical modification techniques that detect unpaired nucleotides in a pool of RNA molecules. Subsequent sequencing of the RNAs reveals the patterns of modified and unmodified bases (their unpaired or paired statuses) and thus indicates how each RNA molecule folds. One of these transcriptome-wide techniques, however, only targets ...
