Hydroxyl radical cleavage of nucleic acids: understanding RNA cleavage profiles and identifying DNA structural motifs
Azad, Robert Navid
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High-resolution techniques to characterize the three-dimensional structure of nucleic acids are critical for understanding the mechanisms of action of biologically important RNA and DNA molecules. Methods based on chemical probing have been particularly useful in gaining insight into the structures of nucleic acids in solution. The hydroxyl radical has been widely adopted as a chemical probe for DNA and RNA structure since its first application to protein-DNA footprinting. This dissertation describes efforts to improve upon the current model of how the hydroxyl radical cleaves the RNA backbone, through the use of specifically deuterated ribonucleoside triphosphates (NTPs). The synthesis and purification of deuterated NTPs are described in detail, as well as their application to the study of two RNAs: the sarcin-ricin loop (SRL) RNA - a biologically active region of ribosomal RNA - and a short RNA designed to lack secondary structure. Measurement of deuterium kinetic isotope effects (KIEs) on the cleavage of these RNAs suggests that it is possible to use this experiment to identify the GUA base triple structural motif that is commonly found in RNA. Abstraction of a 5' ribose hydrogen atom in RNA yields a fragment containing a 5'-aldehyde terminus with the sugar and base intact. Comparison of primer extension products of cleaved SRL RNA with or without deuterium substituted at the C5' ribose position of uracil residues demonstrated that the 5' aldehyde-terminated fragment can serve as a template for reverse transcription. Implications of the presence of a 5'-aldehyde terminus on hydroxyl radical cleavage analysis are discussed in the context of reverse transcriptase-mediated primer extension, a commonly used method. Structural features of naked DNA molecules with known protein binding sequences were explored using hydroxyl radical cleavage analyzed by capillary gel electrophoresis. An application was written in MATLAB to deconvolute and integrate cleavage intensities of hundreds of peaks in an electropherogram. In many cases, comparison of the cleavage profile to the minor groove width found in an X-ray co-crystal structure of the DNA-protein complex revealed a high degree of correlation.