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Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

ABSTRACT: Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target site. Selecting an accessible binding site is made difficult by RNA folding and protein binding in vivo and the need to bind two probes. Even more problematic, has been a lack of methods for efficient cytoplasmic delivery of the probes that would be suitable for eventual applications in vivo in animals. Herein we report the imaging of iNOS mRNA expression in live mouse macrophage cells with PNA·DNA binary FRET probes delivered by a cationic shell crosslinked knedel-like nanoparticle (cSCK). We first demonstrate that FRET can be observed on in vitro transcribed mRNA with both the PNA probes and the PNA·DNA hybrid probes. We then demonstrate that the FRET signal can be observed in live cells when the hybrid probes are transfected with the cSCK, and that the strength of the FRET signal is sequence specific and depends on the mRNA expression level.

SUBMITTER: Wang Z

PROVIDER: S-EPMC3687806 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

Wang Zhenghui Z Zhang Ke K Shen Yuefei Y Smith Jillian J Bloch Sharon S Achilefu Samuel S Wooley Karen L KL Taylor John-Stephen JS

Organic & biomolecular chemistry 20130328 19

Optical imaging of gene expression through the use of fluorescent antisense probes targeted to the mRNA has been an area of great interest. The main obstacles to developing highly sensitive antisense fluorescent imaging agents have been the inefficient intracellular delivery of the probes and high background signal from unbound probes. Binary antisense probes have shown great promise as mRNA imaging agents because a signal can only occur if both probes are bound simultaneously to the mRNA target ...[more]

PMID: 23538604

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Project description:Recently, the functionalization of nanoparticles, either within themselves or on the outer surface and its application in medicine, turned out to be the ultimate goal of nanotechnology. By providing these nanoparticles with chemical functional groups, one can force the nanoparticles to target the markers of the particular diseases or to measure the quantity and distribution of various intracellular species. In this paper, we report our development of a pH-responsive nanocomposite based on lanthanide-doped upconverting nanoparticles (UCNPs). Through multiphoton absorption and energy migration between spatially separated Nd3+, Yb3+, and Tm3+ in a three-layered NaYF4 host coated with FITC (fluorescein-5-isothiocyanate), this nanocomposite can measure the pH with high sensitivity. The fundamental acidity measurement is based on the pH-dependent equilibrium of the bright and dark states of FITC. The tremendous advantages of this system, regarding the pH measurement, come from the fact that the versatility of UCNP-imaging can fully be exploited. This includes the fact that (a) the optical wavelengths for the sensitization (980 nm and/or 808 nm) and the emission bands (UV, visible) are well separated, (b) the spectral overlap between FITC (absorption) and Tm3+ (emission) is substantially high, (c) there is no background signal due to the near-infrared laser, and (d) the signals are consistent regardless of the fluctuations by monitoring the ratio of blue band with respect to the unaffected self-reference (red and near-infrared bands). Moreover, the double shell structure is obviously superior to the core-shell structure in that it enhances the spectral separation between the sensitizer and the emitter in the upconversion process, inhibiting any unnecessary contamination in the spectra. Finally, it is noteworthy that Yb3+ plays crucial roles as a sensitizer at 980 nm excitation and a bridge above which 808 nm excitation migrates from Nd3+ to Tm3+ via the Yb3+ excited state.

Dataset Information

Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

Publications

Imaging mRNA expression levels in living cells with PNA·DNA binary FRET probes delivered by cationic shell-crosslinked nanoparticles.

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