Characterization of DNA and RNA by MALDI-TOF mass spectrometry

DNA and RNA control the transfer of cellular information through many cellular processes. These processes are controlled by the association of polynucleic acids with other biomolecules or with each other. The structures and associations are defined by sequence, number of strands or attachment of another biomolecule. In order to understand these interactions, the binding regions and base modifications must be identified through fast, reliable methods that can be applied to picomolar amounts of sample. Base modifications and higher order structures can be identified by both chemical and enzymatic reactions that specifically degrade particular regions of the polynucleotide leaving only the regions of interest for analysis. Coupling these reactions to matrix assisted laser desorption/ionization (MALDI) mass spectrometry for product analysis provides an efficient and accurate method to detect and locate associations and modifications.; This dissertation focuses on the characterization of DNA and RNA using MALDI mass spectrometry. Special sample preparations were developed to enable the coupling of enzymatic reactions to MALDI. Starting with basic non-covalent associations of DNA only, double stranded regions of DNA were identified by selectively digesting the single stranded regions. Using single strand specific exo- and endonucleases, DNA overhangs and loops were identified. This approach was applied to the study of a non-covalent interaction between an RNA hairpin and peptide. Selective digestion of the single stranded loop and the unbound regions identified a contact point within the hairpin loop of the RNA.; Other investigations were performed where the primary structure of DNA was explored. A specific guanine base modification was produced by intense UV irradiation and identified from a mixture of modified and wild type DNA by identifying the products from chemical degradation of the DNA with MALDI. The mass shift gave insight as to the type of modification present while the products of chemical hydrolysis led to the location of the modification. The expected product, 8-oxoguanine, was not formed in solution as expected. A similar product with an epoxide ring between the C4 and C 5 positions in guanine is proposed. This alternative structure is more likely due to its mass and reactivity in base.