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.

Post-nuclear gene delivery events for transgene expression by biocleavable polyrotaxanes

“…This tendency was also observed in a previous study, in which 14DMAE and 29DMAE were employed as an artificial condenser [11]. Additionally, we showed that the cationic density in DMAE-SS-PRX has a positive influence on the efficiency of DNA release in the nucleus in living cells [10], suggesting that the conditions used in the in vitro experiment would reflect the condition of the nucleus. In this study, we used condensed pDNA particles at an N/P ratio of 5 in all of the DMAE-SS-PRXs.…”

“…In vitro DNA release experiments showed that pDNA was efficiently released from the condensed pDNA particles, when DMAE-SS-PRX with a high cationic density was used [11]. Moreover, we provide evidence to show that cationic density in DMAE-SS-PRX clearly has a positive influence on the efficiency of DNA release in the nucleus in living cells, resulting in high gene expression, as evidenced by imaging the nuclear condensation/decondensation status of pDNA using a new technology [10] Based on the previous results, we hypothesized that a high cationic density in DMAE-SS-PRX would aid nuclear DNA release, resulting in strong transgene expression. In this study, we first screened the optimal DMAE-SS-PRX for nuclear DNA release.…”

“…We previously demonstrated the existence of a close relationship between the efficiency of DNA release and the transfection activity, using an integrated system of R8-MEND and DMAE-SS-PRX [10,11]. It is particularly noteworthy that, from the standpoint of transfection, our integration system, which is comprised of the R8-MEND with DMAE-SS-PRX, was 5-fold greater than that for a conventional R8-MEND with protamine, which is a natural DNA condenser that shows a high transfection activity [18][19][20].…”

“…To date, we reported on the development of an innovative gene delivery system [10,11], prepared by integrating a multifunctional envelope-type nano device (MEND) [5,[12][13][14] and a biocleavable polyrotaxane (DMAE-SS-PRX) [15][16][17] (Fig. 1B).…”

“Programmed packaging” for gene delivery

“…Furthermore, some efforts directed to the use of the NLS failed to enhance the transfection efficacy in JAWS II cells or primary mouse bone marrow-derived dendritic cells (BMDCs). As an alternate approach, we attempted to overcome the nuclear membrane as well as the endosomal membrane via step-wise membrane fusion [68,69]. In this strategy, we developed a tetra-lamellar MEND (T-MEND), in which the pDNA/polycation complex particle was coated with nucleus-fusogenic lipid (composed of caldiolipin and DOPE) and an endosome-fusogenic lipid (composed of phosphatidic acid and DOPE).…”