Inosine (I) is a nucleoside that is a result of the deamination of adenosine (A). It is ubiquitous in nature, due in part to its role in A-to-I editing. There is also damage that is generated by reactive oxygen species (ROS) in RNA and DNA. This can potentially lead to the oxidation of guanosine (G) and I, and this forms their corresponding 8-oxo-7,8-dihydropurines, 8-oxoG and 8-oxoI. These types of lesions could affect enzyme activity for reactions like reverse transcription (RTn) ultimately altering hydrogen bond patterns and base pairing properties. In this work, the thermal denaturation transitions (Tm) of these RNA nucleobases (X=G, I, 8-oxoG, and 8-oxoI) were established via circular dichroism (CD). Reverse transcription and steady-state kinetics were measured, and all these methods revealed strong evidence on the effects that these damaged nucleobases have on the enzyme catalyzed base incorporations and transcription of cDNA. All these experiments used RNA templates that are 29-nt long, and DNA primers that are 17-nt and 18-nt long. The RNA template containing inosine was able to form stable bonds to all four dNTPs (dATP, dCTP, dGTP, and dTTP). In addition, full cDNA synthesis was achieved for the RNA templates containing I, 8-oxoI, and 8-oxoG when hybridized to a DNA primer with cytosine (C) or A at the 18th position. Through reverse transcription the binding patterns that are formed reveal how the lesions are altering enzymatic activity, and this data can demonstrate how the structure is impacted when RNA bases are damaged through ROS and deamination.
|Advisor:||Resendiz, Marino J. E.|
|Commitee:||Ren, Xiaojun, Knight, Jefferson|
|School:||University of Colorado at Denver|
|School Location:||United States -- Colorado|
|Source:||MAI 81/12(E), Masters Abstracts International|
|Keywords:||Guanosine, Inosine, 8-oxoG, 8-oxoI, Dihydropurine|
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