Imaging mRNA Expression in Live Cells via PNA·DNA Strand Displacement-Activated ProbesReport as inadecuate




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Journal of Nucleic AcidsVolume 2012 2012, Article ID 962652, 11 pages

Research Article

Department of Chemistry, Washington University, St. Louis, MO 63130, USA

Department of Chemistry, Texas A&M University, P.O. Box 30012, College Station, TX 77842-3012, USA

Received 16 June 2012; Accepted 30 July 2012

Academic Editor: Eriks Rozners

Copyright © 2012 Zhenghui Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 -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.





Author: Zhenghui Wang, Ke Zhang, Karen L. Wooley, and John-Stephen Taylor

Source: https://www.hindawi.com/



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