Molecular imaging of gene expression at the level of RNA in living animals.

摘要

The ability to image gene expression in living subjects noninvasively and repetitively has the potential to improve clinical diagnosis and management of disease, as well as inform researchers about fundamental biological processes. By targeting gene expression at the level of RNA, imaging probes can potentially be designed that are highly specific and generalizable. This task has traditionally been accomplished using radiolabeled antisense oligonucleotides (RASONs). However, RASONs have certain drawbacks including lack of an amplifiable and activatable signal, as well as poor delivery across cellular membranes. This dissertation focused on developing novel strategies for imaging RNA that help to overcome these limitations. The first strategy discussed features spliceosome-mediated RNA trans-splicing (SMaRT). Originally developed for therapeutic gene therapy to replace defective exons, the experiments reported here demonstrate that SMaRT technology can be used to trans- splice a reporter gene into a target pre-mRNA of interest. The reporter gene is translated if and only if it first trans-splices into the target and thus the signal reflects the level of expression of the target gene. Studies revealed factors influencing trans-splicing specificity and efficiency, and a proof of principle experiment was performed to show that the trans-splicing reaction could be detected in living animals. The second strategy reported involves dual protein-oligonucleotide conjugates. This approach is similar in design to RASONs except that it uses two interacting proteins that require binding of both antisense molecules to the same target RNA in the correct orientation in order to produce a signal. It was originally thought that split reporter proteins could be used as the interacting proteins, but after it was found that the protein halves did not express well, the strategy was transitioned to use bioluminescence resonance energy transfer (BRET). By using BRET, I was able to demonstrate the validity of the strategy and define its specificity and sensitivity using a solution-based assay. Both of these projects demonstrate that unorthodox approaches to RNA-based probes have significant potential for the future of gene expression imaging at the RNA level.