title: 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.
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130, USA.
authors.forename: Zhenghui
authors.initials: Z
authors.lastname: Wang
authors.forename: Ke
authors.initials: K
authors.lastname: Zhang
authors.forename: Yuefei
authors.initials: Y
authors.lastname: Shen
authors.forename: Jillian
authors.initials: J
authors.lastname: Smith
authors.forename: Sharon
authors.initials: S
authors.lastname: Bloch
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
authors.forename: Karen L
authors.initials: KL
authors.lastname: Wooley
authors.forename: John-Stephen
authors.initials: JS
authors.lastname: Taylor
mesh_terms: D000818:Animals; D002412:Cations / Q000737:chemistry; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D016503:Drug Delivery Systems; D031541:Fluorescence Resonance Energy Transfer; D008264:Macrophages / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D015394:Molecular Structure; D053758:Nanoparticles / Q000737:chemistry; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D020135:Peptide Nucleic Acids / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics*
chemical_list: D002412:Cations; D003432:Cross-Linking Reagents; D015345:Oligonucleotide Probes; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D004247:DNA; D052247:Nitric Oxide Synthase Type II
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