Application of FRET Technology to the In Vivo Evaluation of Therapeutic Nucleic Acids (ANTs)

Abstract

Developing applications for therapeutic nucleic acids (TNAs) (i.e. ribozymes, antisense oligodeoxynucleotides (AS‐ODNs), siRNA and aptamers) requires a reporter system designed to rapidly evaluate their in vivo effect. To this end we designed a reporter system based on the fluorescence resonance energy transfer (FRET) engineered to release the FRET effect produced by two green fluorescent protein (GFP) variants linked by a TNA target site. Because the FRET effect occurs instantaneously when two fluorophores are very close to each other (>100nm) stimulating emission of the acceptor fluorophore by the excitation of the donor fluorophore it has been widely use to reveal interactions between molecules. The present system (FRET2) correlates the FRET effect with the in vivo activity of distinct types of TNAs based on a model consisting of RNA from human papillomavirus type 16 (HPV‐16) previously shown accessible to TNAs. HPV‐16 is the most common papillomavirus associated with cervical cancer, the leading cause of death by cancer in Mexico. The FRET2 system was first tested in vitro and then used in bacteria in which transcription is linked to translation allowing controlled expression and rapid evaluation of the FRET2 protein. To assure accessibility of the target mRNA to TNAs, the FRET2 mRNA was probed by RNaseH assays prior FRET testing. The fluorescence features of the FRET2 system was tested with different FRET‐producing GFP donor‐acceptor pairs leading to selection of green (donor) and yellow (acceptor) variants of GFP as the most efficient. Modifications in aminoacid composition and linker length of the target sequence did not affect FRET efficiency. In vivo AS‐ODN‐mediated destruction of the chimerical FRET2 reporter mRNA resulted in the recovery of GFP fluorescent spectrum in a concentration and time dependent manner. Reported anti‐HPV ribozymes were also tested with similar results. Therefore, we conclude that the FRET effect can be a useful tool in the development of TNAs.