CHARACTERIZATION OF A GTP-BINDING DNA APTAMER

Abstract

<p>Aptamers are single-stranded nucleic acids that can bind a wide variety of targets. The target-binding events involved structural changes of an aptamers and have been exploited to create fluorescent reporters. Aptamer-based fluorescent sensors are highly sensitive and selective and can be adapted as signaling components in detection assays. Previously, an aptamer that binds GTP was isolated by in vitro selection from a random-sequence DNA library. Guanosine-5’-triphosphates (GTP) is an important biological cofactor that is widely utilized by many biological receptors and enzymes. Assays that monitor these receptors and enzymes, such as radioactive binding assays using GTP analog [35S] GTPγS, are not homogeneous and required extensive sample treatments. In contrast, fluorescent reporters made using functional nucleic acids, such as aptamers, are convenient to use and provide real-time detection for a plethora of targets. If the latter detection method could be applied for the detection of GTP, this would provide a more simple and convenient alternative to current methods.</p> <p>In this thesis, we explore the possibility of designing a novel assay that unifies aptamer characterization with structure-switching. We used this assay to analyze the sequence requirement and recognition specificity of this GTP-binding DNA aptamer. And, we found that binding activity can be retained with up to 40% of nucleotides removed and the sequence identity can be simplified to only guanine and thymine residues. The sequence information about this aptamer can facilitate the development of an efficient fluorescence-signaling aptamer for real-time detection of GTP.</p>