Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes

Abstract

Probes for monitoring mRNA expression in vivo are of great interest for the study of biological and biomedical problems, but progress has been hampered by poor signal to noise and effective means for delivering the probes into live cells. Herein we report a PNA·DNA strand displacement-activated fluorescent probe that can image the expression of iNOS (inducible nitric oxide synthase) mRNA, a marker of inflammation. The probe consists of a fluorescein labeled antisense PNA annealed to a shorter DABCYLplus-labeled DNA which quenches the fluorescence, but when the quencher strand is displaced by the target mRNA the fluorescence is restored. DNA was used for the quencher strand to facilitate electrostatic binding of the otherwise netural PNA strand to a cationic shell crosslinked knedel-like (cSCK) nanoparticle which can deliver the PNA·DNA duplex probe into cells with less toxicity and greater efficiency than other transfection agents. RAW 264.7 mouse macrophage cells transfected with the iNOS PNA·DNA probe via the cSCK showed a 16 to 54-fold increase in average fluorescence per cell upon iNOS stimulation. The increase was 4 to 7-fold higher than that for a non-complementary probe, thereby validating the ability of a PNA·DNA strand displacement-activated probe to image mRNA expression in vivo.

Nanotechnology for delivery of peptide nucleic acids (PNAs)

“…In another study, cSCK nanoparticles were used to deliver PNA-based strand displacement probes for live-cell imaging of iNOS mRNA [39]. Subsequently, this cSCK nanoparticle was used for delivery of antisense PNA.DNA binary FRET probes for both in vitro and in vivo imaging [38].…”

Imaging Genetic Information

“…13.11) [145]. This NP consisted of a polystyrene core and a poly-N-2-aminoethylacrylamide shell, which is largely protonated at pH 7, though a number of amines remain unprotonated [146].…”

Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection

“…The displaced or spontaneously dissociated ODN is also expected to be enzymatically degraded thereby ensuring binding of the PNA to the target mRNA. This strand displacement strategy has recently been successfully used to generate fluorescent imaging probes for iNOS mRNA [19].…”

“…The results can be understood by considering the amount of iNOS mRNA present in the cell compared with the amount of probe. As part of another study [19], we had determined, by absolute RT-PCR using authentic iNOS mRNA to generate a standard curve, that the iNOS level rises 100-fold 18 h after induction, to about 76 000 copies of iNOS mRNAs per cell. Given that 10 , there would be about 1200-, 235-and 23-fold excess PNA probe over target mRNA.…”

“…Studies that show direct and specific targeting of iNOS mRNA both in vitro and in vivo are limited. Our work and that of others have shown that peptide nucleic acid (PNA) has a number of useful properties that make it an ideal choice for targeting candidate mRNA [13][14][15][16][17][18][19].…”