Imaging Target mRNA and siRNA‐Mediated Gene Silencing In Vivo with Ribozyme‐Based Reporters

Abstract

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.

Molecular Probes for Bioluminescence Imaging

“…The engineered ribozyme-based reporter system, which employs antisense RNA binding and reporter gene system including -lactamase and FLuc, was proved to an efficient tool in imaging mutant p53 mRNA both in single cells and noninvasively in living mice. Compared with previous methods for mRNA imaging, this system shows high sensitivity for target detection and excellent compatibility with many in vivo noninvasive imaging modalities [29]. …”

Molecular MR Imaging of Cancer Gene Therapy: Ferritin Transgene Reporter Takes the Stage

“…Recently, a new type of direct imaging has been proposed that uses transsplicing ribozyme. 24,25 Reporter gene imaging, categorized as indirect, is widely used for imaging gene expression in vivo, and various modality-speciˆc reporter genes have been described (Table 2). 1,26 Complementary imaging probes developed by coupling with radioisotope, paramagnetic ion, or ‰uorescence can detect reporter transgene expression.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…412 The enzymatic activity of the Tetrahymena ribozyme has been modified to develop an RNA imaging tool, using antisense RNA binding and reporter gene systems for target specificity and signal amplification. 413 RNA oligonucleotides (40-60nt) are able to selfassemble into versions of the Azoarcus ribozyme, in which an internal guide sequence interacts with a complementary triplet CAU. Mutagenesis of the CAU sequence have been used to determine the self-assembly efficiency, with CAC retaining the greatest activity, GAU the least.…”