| Description |
Reactive oxygen species can target nucleobases of DNA and RNA. One of the major products of oxidation of nucleic acids by ROS is 8-oxo-7,8-dihydroguanine (OG) which is prone to further oxidation to yield the two hydantoin lesions, 5-guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp). These three lesions are mutagenic and their occurrence can significantly affect normal cell functions or cell survival. The OG, Gh, and Sp are thus removed from DNA and RNA and then are excreted from the cells in the form of free nucleobases. Concentrations of these species increase during oxidative stress and can be used as an indicator of this condition. The first part of this dissertation is devoted to development of aptamer-based sensors that can be used for detection of Gh in the free base form. We described synthesis of Gh and used an attachment-free SELEX method for in vitro selection of aptamers for the hydantoin. Recent studies have shown that OG can play a regulatory role in both DNA and RNA. However, if oxidation of guanine in nucleic acids plays a regulatory role, it has to be directed toward specific sites. Testing whether this is true can be greatly facilitated by mapping positions of oxidation sites. The second part of this dissertation is focused on investigating whether sequencing of cDNA after reverse transcription (RT) of a target RNA can be used for mapping oxidation sites. In order to do so, we first studied the composition of canonical bases that are inserted opposite OG, Gh, and Sp in RNA by RT enzymes. We have found that SuperScript III is capable of efficiently bypassing the studied lesions resulting in insertion of a mixture of A and C opposite OG and mixture of A and G opposite Gh and Sp. These data indicated that reverse transcription of oxidized bases is a viable approach for mapping their positions in RNA. Additionally, we have completed the work required to quantify the mutation signature of oxidized lesions using Illumina sequencing and studied the dependence of the efficiency of deletion bypass of hydantoin lesions on the sequence context. |
