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Published on: August 6, 2014
Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated Probes
Zhenghui Wang1, Ke Zhang, Karen L Wooley
1Department of Chemistry, Washington University, St. Louis, MO 63130, USA.
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 DABCYL(plus)-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.
Insights
Researchers developed a novel PNA·DNA fluorescent probe to image inflammation marker iNOS mRNA in vivo. This probe, delivered by cSCK nanoparticles, shows significant fluorescence enhancement, enabling effective mRNA expression monitoring in cells.
Area of Science:
- Molecular Biology
- Biomedical Imaging
- Nanotechnology
Background:
- In vivo mRNA expression monitoring is crucial for biological and biomedical research.
- Existing probes suffer from poor signal-to-noise ratios and inefficient cellular delivery.
- Inducible nitric oxide synthase (iNOS) mRNA is a key marker for inflammation.
Purpose of the Study:
- To develop and validate a PNA·DNA strand displacement-activated fluorescent probe for imaging iNOS mRNA expression.
- To assess the probe's efficacy and safety for in vivo cellular delivery using cSCK nanoparticles.
- To evaluate the probe's ability to accurately quantify iNOS mRNA levels in response to inflammatory stimuli.
Main Methods:
- Design of a PNA·DNA duplex probe utilizing fluorescence quenching and strand displacement.
- Conjugation of the PNA·DNA probe to cationic shell crosslinked knedel-like (cSCK) nanoparticles for enhanced cellular delivery.
- Transfection of RAW 264.7 mouse macrophage cells with the probe-nanoparticle complex.
- Stimulation of iNOS expression and subsequent fluorescence imaging to quantify mRNA levels.
Main Results:
- The PNA·DNA probe demonstrated successful imaging of iNOS mRNA expression in stimulated macrophage cells.
- Cells transfected with the probe via cSCK nanoparticles showed a 16 to 54-fold increase in average fluorescence.
- The observed fluorescence increase was significantly higher (4 to 7-fold) compared to a non-complementary control probe, validating specificity.
Conclusions:
- PNA·DNA strand displacement-activated probes offer a sensitive and specific method for in vivo mRNA imaging.
- cSCK nanoparticles provide an efficient and low-toxicity platform for delivering nucleic acid probes into cells.
- This technology holds promise for advancing the study of inflammatory processes and other biomedical applications requiring mRNA monitoring.
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