Health & Environmental Research Online (HERO)


Print Feedback Export to File
2966986 
Journal Article 
Chemical probing of RNA with the hydroxyl radical at single-atom resolution 
Ingle, S; Azad, RN; Jain, SS; Tullius, TD 
2014 
1 
Nucleic Acids Research
ISSN: 0305-1048
EISSN: 1362-4962 
42 
20 
12758-12767 
English 
25313156 
While hydroxyl radical cleavage is widely used to map RNA tertiary structure, lack of mechanistic understanding of strand break formation limits the degree of structural insight that can be obtained from this experiment. Here, we determine how individual ribose hydrogens of sarcin/ricin loop RNA participate in strand cleavage. We find that substituting deuterium for hydrogen at a ribose 5'-carbon produces a kinetic isotope effect on cleavage; the major cleavage product is an RNA strand terminated by a 5'-aldehyde. We conclude that hydroxyl radical abstracts a 5'-hydrogen atom, leading to RNA strand cleavage. We used this approach to obtain structural information for a GUA base triple, a common tertiary structural feature of RNA. Cleavage at U exhibits a large 5' deuterium kinetic isotope effect, a potential signature of a base triple. Others had noted a ribose-phosphate hydrogen bond involving the G 2'-OH and the U phosphate of the GUA triple, and suggested that this hydrogen bond contributes to backbone rigidity. Substituting deoxyguanosine for G, to eliminate this hydrogen bond, results in a substantial decrease in cleavage at G and U of the triple. We conclude that this hydrogen bond is a linchpin of backbone structure around the triple.