Book of Abstracts: Albany 2005
Deuterium Kinetic Isotope Effect Experiments on RNA Cleavage by the Hydroxyl Radical
Reactive oxygen species are known to cause non-specific damage to nucleic acids. One of the most common reactive oxygen species, the hydroxyl radical (•OH), abstracts a hydrogen atom from a ribose sugar in the backbone of DNA and RNA, leading to strand cleavage. Numerous studies have employed •OH as a high-resolution chemical probe of the structure of DNA. More recently the hydroxyl radical has become widely used as a high-resolution probe of RNA folding and structure. Little is known, though, about the detailed chemical mechanism of RNA cleavage by the hydroxyl radical. In order to investigate the mechanism of RNA strand cleavage by •OH, we have used chemical and enzymatic synthesis to incorporate deuterium selectively at the 5' and 5", 4', 3' and 2' positions of the ribose sugars of both GTP and ATP. We then used in vitro transcription, with either 5'-dideuterated, 4' deuterated, 3' deuterated, or 2' deuterated GTP or ATP, along with the other three ribonucleoside triphosphates, to prepare a 29mer analog of the Sarcin-Ricin loop, a key structural feature of 28S ribosomal RNA. We report here the first observation of a deuterium kinetic isotope effect on •OH cleavage of RNA. These experiments provide the first direct evidence for the mechanism of RNA cleavage by the hydroxyl radical. We expect that these and future results using this experimental system will be useful in developing a more detailed interpretation of RNA structure based on hydroxyl radical cleavage experiments.
Department of Chemistry