The Characterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry.
Date of Award
Doctor of Philosophy (PhD)
Patrick A. Limbach
The use of mass spectrometry for the characterization of nucleic acids is presented in this work. One area of research was aimed at developing a method for the characterization of pseudouridine (Psi) containing samples derivatized with N-cyclohexyl-N' -beta-(4-methylmorpholinium)methylcarbodiimide (CMC). These experiments consisted of the derivatization of a tRNA molecule with CMC followed by analysis of the sample using polyacrylamide gel electrophoresis and mass spectrometry. Mass spectrometric analysis of the sample revealed that the derivatization of the sample was occurring to generate a species with one CMC group. This data is significant because it demonstrates that the comparably small mass shift caused by the presence of the CMC group can be distinguished in an intact nucleic acid sample which has been exposed to high salt conditions that typically degrade mass spectrometric data. Another area of research demonstrates the first example of mass spectrometry to determine the specificity of RNase H cleavage reactions of oligonucleotides. In this case, an oligoribonucleotide hairpin was subjected to RNase H cleavage using chimeric oligonucleotides, and the resulting samples were analyzed by mass spectrometry. The experimentally determined mass-to-charge (m/z) values were compared to those predicted to determine that selective cleavage did indeed occur using the reaction conditions described. Finally, the reaction and purification conditions required to mass spectrometry are presented. It was found that the isolation of a large region (534-mer) was more efficient than the isolation of a smaller region (39-mer) due to the difficulties involved in separating such a small sample from the comparably larger fragments which also result during RNase H cleavage. In addition, the mass spectrometric characterization of the RNase T1 digests of the isolated region to determine if the correct region is being isolated, as well as to determine the specificity of the reaction, is presented. Although it appears that non-specific cleavage is occurring, a large number of the expected m/z values for the fragments of the desired region are observed, indicating that this region may be present in the isolated product.
Polo, Lenore Marie, "The Characterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry." (1999). LSU Historical Dissertations and Theses. 6923.