title: Quantitative analysis of condensation/decondensation status of pDNA in the nuclear sub-domains by QD-FRET.
abstract: Recent studies indicate that controlling the nuclear decondensation and intra-nuclear localization of plasmid DNA (pDNA) would result in an increased transfection efficiency. In the present study, we established a technology for imaging the nuclear condensation/decondensation status of pDNA in nuclear subdomains using fluorescence resonance energy transfer (FRET) between quantum dot (QD)-labeled pDNA as donor, and rhodamine-labeled polycations as acceptor. The FRET-occurring pDNA/polycation particle was encapsulated in a nuclear delivery system; a tetra-lamellar multifunctional envelope-type nano device (T-MEND), designed to overcome the endosomal membrane and nuclear membrane via step-wise fusion. Nuclear subdomains (i.e. heterochromatin and euchromatin) were distinguished by Hoechst33342 staining. Thereafter, Z-series of confocal images were captured by confocal laser scanning microscopy. pDNA in condensation/decondensation status in heterochromatin or euchromatin were quantified based on the pixel area of the signals derived from the QD and rhodamine. The results obtained indicate that modulation of the supra-molecular structure of polyrotaxane (DMAE-ss-PRX), a condenser that is cleaved in a reductive environment, conferred euchromatin-preferred decondensation. This represents the first demonstration of the successful control of condensation/decondensation in specific nuclear sub-domain via the use of an artificial DNA condenser.
authors.affiliation: Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan.
authors.forename: Sharif M
authors.initials: SM
authors.lastname: Shaheen
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Akita
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamashita
authors.forename: Ryo
authors.initials: R
authors.lastname: Katoono
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Yui
authors.forename: Vasudevanpillai
authors.initials: V
authors.lastname: Biju
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Hideyoshi
authors.initials: H
authors.lastname: Harashima
mesh_terms: D019921:Biotinylation; D002467:Cell Nucleus / Q000235:genetics*; D003505:Cyclodextrins / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D008156:Luciferases / Q000235:genetics; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics*; D020442:Poloxamer / Q000737:chemistry; D045663:Quantum Dots; D043862:Rotaxanes / Q000737:chemistry; D019076:Transgenes
chemical_list: D003505:Cyclodextrins; D043862:Rotaxanes; C475673:polyrotaxane; D020442:Poloxamer; D004247:DNA; D008156:Luciferases