Study of RNA structure by affinity cleaving

Citation

Han, Hogyu (1995) Study of RNA structure by affinity cleaving. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/fwfk-4p70. https://resolver.caltech.edu/CaltechETD:etd-10052007-081104

Abstract

NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Chapter 1. Sequence-specific recognition of double helical RNA and RNA•DNA by triple helix formation. The stabilities of eight triple helical pyrimidine•purine•pyrimidine structures comprised of identical sequence but different RNA (R) or DNA (D) strand combinations were measured by quantitative affinity cleavage titration. The differences in equilibrium binding affinities reveal the importance of strand composition. For the sequences studied here, the stabilities of complexes containing a pyrimidine third strand D or R and purine•pyrimidine double helical DD, DR, RD, and RR decrease in order: D + DD, R + DD, R + DR, D + DR > R + RD,R + RR >> D + RR,D + RD (pH 7.0, 25°C, 100 mM NaCl / 1mM spermine). These findings suggest that RNA and DNA oligonucleotides will be useful for targeting (i) double helical DNA and (ii) RNA•DNA hybrids if the purine Watson-Crick strand is DNA. However, RNA, but not DNA, oligonucleotides will be useful for sequence-specific binding of (i) double helical RNA and (ii) RNA•DNA hybrids if the purine Watson-Crick strand is RNA. This has implications for the design of artificial ligands targeted to specific sequences of double helical RNA and RNA•DNA hybrids. Chapter 2. Different conformational families of pyrimidine•purine•pyrimidine triple helices depending on backbone composition. Different helical conformations of DNA (D), RNA (R), and DNA•RNA (DR) hybrid double and triple helices have been detected using affinity cleavage analysis. Synthetic methods were developed to attach EDTA•Fe to a single nucleotide on RNA as well as DNA oligonucleotides. Cleavage patterns generated by a localized diffusible oxidant in the major groove on the pyrimidine strand of four purine•pyrimidine double helices consisting of all DNA, all RNA, and the corresponding hybrids reveal that the relative cleavage intensity shifts to the 5' end of the purine strand increasingly in the order: DD<DR<RD<RR. These results are consistent with models derived from structural studies. In six pyrimidine•purine•pyrimidine triple helices, the altered cleavage patterns of the Watson-Crick pyrimidine strands reveal at least two conformational families: (i) D+DD, R+DD, D+DR, and R+DR and (ii) R+RD and R+RR. Chapter 3. Visualization of RNA tertiary structure by RNA-EDTA•Fe(II) autocleavage. Analysis of tRNA[...]•Fe(II). To test whether intramolecular autocleavage of RNA labeled at a single nucleotide position with EDTA•Fe(II) is a useful approach for the investigation of tertiary structures of RNAs, yeast phenylalanine tRNA-EDTA was synthesized by the incorporation of uridine-EDTA (*U) at position U47. Autocleavage of tRNA[...] in the presence of Fe(NH4)2(SO4)2 and dithiothreitol produced a set of cleavage fragments which are in general agreement with the three-dimensional structure derived from x-ray analysis. Chapter 4. Mapping regions in eukaryotic ribosomes that are accessible to methidiumpropyl-EDTA•Fe(II) and EDTA•Fe(II). Methidiumpropyl-EDTA•Fe(II) (MPE•Fe(II)) and EDTA•Fe(II) were used to investigate the structure of Drosophila melanogaster ribosomes. Cleavage reactions were performed on intact ribosomes in cell lysates in vitro and analyzed by primer extension with reverse transcriptase using oligodeoxynucleotide primers. Regions of 18S and 28S ribosomal RNAs (rRNAs) which are accessible to MPE•Fe(II) and EDTA•Fe(II) are located almost exclusively within expansion segments. The accessibility of these regions to cleavage indicates that they are exposed on the surface of eukaryotic ribosomes. These results provide valuable information about the overall tertiary structure of rRNA in ribosomes.

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