Development of aptamer based targeted reversibly attenuated probes /
Abstract (Summary)The targeted reversely attenuated probe (TRAP) is an aptamer-based biosensor in which the aptamer activity can be regulated by a specific nucleic acid sequence such as an mRNA. The TRAP has the potential of being developed for imaging gene expression in vivo. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regDNA). I developed 8att-cmRas20 and 9att-cmRas15 ATP DNA TRAPs with regDNA-dependent aptamer activity. The results suggested that, as well as inhibiting the aptamer, the attenuator also acted as a structural guide, much like a chaperone, to promote proper folding of the TRAP such that it can be fully activated by the regDNA. We also showed that activation of the aptamer in the TRAP at physiological temperatures by a regDNA complementary to the intervening sequence was sensitive to single base mismatches. I then utilized a tiled microarray to identify the regions on the mRNA that can be easily targeted by antisense oligonucleotides and the results showed that the microarray approach provided a better match with the in vivo antisense than computational analysis. Selected from a microarray study, the 20nt antisense oligonucleotide that targets positions 741-760 of Lcn2 mRNA was chosen for the malachite green (MG) RNA TRAP. To develop the probe, the structure of the 38nt MG RNA aptamer was first destabilized by introducing truncations and mutations. By rational design and after screening different MG RNA TRAPs, the aptamer activity of an TRAP that consists of a 32nt modified MG RNA aptamer and 9nt attenuator sequence was shown to be increased about 20-fold in the presence of a 20nt regDNA. A yeast selection system has been designed to test the function in vivo of the selected TRAP. A hammerhead (HH) ribozyme - aptamer/TRAP - hepatitis Delta Virus (HDV) ribozyme cassette was constructed to produce aptamers or TRAPs with defined and homogeneous 5' and 3' ends.
School:Iowa State University
School Location:USA - Iowa
Source Type:Master's Thesis
Date of Publication: