Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA–peptide chimeras

Lundin, Karin E.; Ge, Rongbin; Svahn, Mathias G.; Törnquist, Elisabeth; Leijon, Mikael; Brandén, Lars J.; Smith, C. I. Edvard

doi:10.1016/j.bioeng.2003.10.003

Cited by 22 publications

(25 citation statements)

References 21 publications

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“…The combined organic extracts were washed with NaOH (aq., 0.2 M, 2 Â 1000 ml) in which the aqueous layers were back extracted with dichloromethane (2 Â 300 ml on the pEGFPLuc plasmid (Clonetech, BD Bioscience, CA, USA) and modified as described previously, was used. 45 It was isolated from grown DH5-á cells according to the Qiagen kit (Qiagen, Chatsworth, CA, USA) protocol and its concentration was estimated using ultraviolet-visible spectroscopy. Formulations of DNA nanoparticles were prepared by equivolumetric mixing of DNA at the desired concentration with fatty acid-spermine conjugates at various concentrations corresponding to different charge ratios (NH 3 +/PO4À), ranging from 0.3:1 to 10:1.…”

Section: Methodsmentioning

confidence: 99%

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

et al. 2009

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The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring moleculesfatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C 16 and C 18 hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl-and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.

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Section: Methodsmentioning

confidence: 99%

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

et al. 2009

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“…One way to facilitate the specific interaction is to let the anchors bind to sites in close proximity to each other. Both ''linear'' PNA (Lundin et al 2004) and bisPNA (Kurakin et al 1998;Lundin et al 2005) show cooperative binding and enhanced labelling of the plasmid, if the target sites are placed within a distance of three to four bases or less. The use of partly overlapping ''opener ''molecules will further facilitate the binding of positively charged PNA-NLS fusions to the correct sites and allow the use of lower concentrations .…”

Section: Increasing Intracellular Transport and Nuclear Transfermentioning

confidence: 98%

“…4 Binding of importin-a to different PNA-NLS constructs hybridized to a 4 BS-plasmid as determined by ELISA. Plasmid DNA was pre-hybridized with PNA-NLS and the hybridization was confirmed by gel analysis as described in (Lundin et al 2004). The DNA was then coated on ELISA-plates and the accessibility of the NLS signal was detected by addition of recombinant importin-a-GST fusion protein.…”

Section: Increasing Intracellular Transport and Nuclear Transfermentioning

confidence: 99%

Nanotechnology approaches for gene transfer

et al. 2009

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In both basic research as well as experimental gene therapy the need to transfer genetic material into a cell is of vital importance. The cellular compartment, which is the target for the genetic material, depends upon application. An siRNA that mediates silencing is preferably delivered to the cytosol while a transgene would need to end up in the nucleus for successful transcription to occur. Furthermore the ability to regulate gene expression has grown substantially since the discovery of RNA interference. In such diverse fields as medical research and agricultural pest control, the capability to alter the genetic output has been a useful tool for pushing the scientific frontiers. This review is focused on nanotechnological approaches to assemble optimised structures of nucleic acid derivatives to facilitate gene delivery as well as promoting down regulation of endogenous genes.

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“…We have used a homolog to the asymmetric cyanine dye thiazole orange (SYBR 103; Molecular Probes, Inc., The agarose gel, 0.8% in 0.25× TBE buffer [32], was running at 100 V for 6 h. (In the lane with unpurified, non-PNA-exposed control plasmid, the amount of DNA is slightly higher than in the other two control lanes; compare also with unpurified, non-PNA-exposed 12-site plasmid.) Reproduced with permission from Elsevier [33]. (B) Plasmid analyzed on agarose gel with chloroquine.…”

Section: Visualizing Pna-nucleic Acid Interactionsmentioning

confidence: 99%

Adding functional entities to plasmids

Svahn

Lundin

et al. 2004

J. Gene Med.

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Non-viral gene therapy constitutes an alternative to the more common use of viral-mediated gene transfer. Most gene transfer methods using naked DNA are based upon non-sequence-specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence-specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long-term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo.

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Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA–peptide chimeras

Cited by 22 publications

References 21 publications

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

Fatty acid–spermine conjugates as DNA carriers for nonviral in vivo gene delivery

Nanotechnology approaches for gene transfer

Adding functional entities to plasmids

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