Date of Award
Master of Science
Theses & dissertations (Interdisciplinary)
Reporter genes, such as luciferase and green fluorescent protein (GFP), have simplified efforts to study transcriptional regulatory elements, tag endogenous proteins, and label and track individual cells in vivo. While these reporters represent a major advance in cellular imaging they have significant limitations. Luciferase, GFP and other reporter genes are restricted by a high cellular energy requirement for their synthesis and a long lag time between the initiation of transcription and reporter protein expression. Also, the method for study of endogenous RNAs utilizing reporter genes is quite complex and limited.
RNA reporters could allow determination of rapid changes in gene expression and may also be used as a tag to make RNA fusion constructs to track other RNAs in vivo. As there is no known naturally produced RNA with inherent fluorescence sufficient for imaging, our approach has been to use RNA aptamers to bind a target molecule with a fluorescent or radioactive label. Aptamers, single stranded RNA or DNA molecules, have high binding affinity and specificity, low molecular mass, and have been selected to recognize enzymes, receptors, growth factors and small molecules.
This work describes part of an effort to develop an RNA tag and reporter gene using the RNA aptamer for theophylline. For the tag to be successful in vivo, the ligand cannot be a natural component of the cell. Theophylline and its aptamer were chosen because theophylline is a small molecule drug and its aptamer has proven functional in bacteria, yeast and cultured human cells.
The proposed IMAGEtag (intracellular multi aptamer genetic tag) will consist of a string of multiple tandem aptamers that can be expressed in cells as RNAs. Multiaptamers were constructed in order to increase the binding capacity of the IMAGEtags. Initially, short multiaptamers were cloned using synthetic oligonucleotides. Elongated multiaptamers were constructed utilizing a modified recursive directional ligation (RDL) technique that employs the non palindromic restriction enzymes BsaI and BsmAI. The multiaptamers were cloned into a mammalian expression vector containing a β-actin promoter and CMV enhancer and expressed in CCL64 cells. Expression and stability of the IMAGEtags were analyzed by real time reverse transcription PCR. The ligands for this IMAGEtag were radioactive theophylline-[3H] and GK38, a theophylline analog labeled with the fluorescent molecule rhodamine-B. GK38 was examined utilizing assays for cellular efflux and localization. Efflux assays were also performed with rhodamine-B, and theophylline-[3H]. Theophylline-[3H] was used to investigate whether IMAGEtag expression had an effect on the cellular concentration of theophylline.
Cloning of a theophylline multiaptamer using synthetic oligonucleotides resulted in a construct with six aptamer repeats, which was used to create constructs with 18, 90 and 270 repeats by the RDL technique. Expression of the 18 repeat multiaptamer in CCL64 cells resulted in a maximum RNA level 24 hours after transfection, and the RNA was determined to have high stability with an approximate half-life of 43 hours. Using a rhodamine-theophylline conjugate to track theophylline in the cells revealed that it binds to endogenous cellular components localized mainly in the Golgi apparatus. Efflux assays showed that 55-60% of the conjugate remains bound to the cells after 1 hour, compared to 30-40% for rhodamine-B and 60-80% for theophylline. It was determined that expression of the theophylline IMAGEtag did not have any effect on the cellular concentration of theophylline. However, it was not determined if the tandemly linked theophylline aptamers in this RNA were capable of binding theophylline.
The observed binding of theophylline and rhodamine-theophylline to cellular components creates a high background signal that obscures a potential signal from the IMAGEtag. This could limit the effectiveness of a reporter system based on theophylline and the theophylline aptamer, and other aptamers and ligands may be more suitable for use as reporters. Alternatively, utilizing Fyrster resonance energy transfer (FRET), a pair of fluorescently labeled aptamer ligands could generate a unique signal when binding to adjacent aptamers in an IMAGEtag.
Hans Eirik Haarberg
Haarberg, Hans Eirik, "Theophylline IMAGEtags (Intracellular Multi Aptamer Genetic tags): the development and evaluation of an RNA reporter system based on the theophylline aptamer" (2009). Graduate Theses and Dissertations. 10578.