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The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases.
- Source :
-
The journal of gene medicine [J Gene Med] 2004 May; Vol. 6 (5), pp. 565-73. - Publication Year :
- 2004
-
Abstract
- Background: Nuclease degradation of plasmid DNA (pDNA) vectors after delivery and during trafficking to the nucleus is a barrier to gene expression. This barrier may be circumvented by shielding the pDNA from the nuclease-rich cell environment with adjuvants or by using nuclease inhibitors. A different alternative that is explored in this work is to make pDNA vectors more nuclease-resistant a priori.<br />Methods and Results: The hypothesis that a significant part of nuclease attack is directed towards certain labile sequences in a pDNA model (pVAX1/lacZ) was first tested. Homopurine-rich tracts in the bovine growth hormone polyadenylation signal (BGH poly A) were identified as labile sequences using S1 nuclease as a probe. Two pDNA variants were then created by replacing the BGH poly A region with the SV40 or a synthetic poly A signal. A study of plasmid degradation in eukaryotic cell lysates and mice plasma showed that the half-life of the supercoiled isoforms of the new vectors was always higher when compared with the control plasmid. An in vitro assay of the reporter beta-galactosidase in transfected CHO cells further showed that gene expression with the new pDNA variants was not affected negatively by the plasmid modifications.<br />Conclusions: The replacement of labile sequences in plasmid DNA vectors improves resistance towards nuclease attack as shown by the increased half-lives of supercoiled plasmid isoforms incubated with endo/lysosomal, cytoplasmatic and blood plasma enzymes.<br /> (Copyright 2004 John Wiley & Sons, Ltd.)
- Subjects :
- Animals
CHO Cells
Cattle
Cricetinae
Cricetulus
Growth Hormone chemistry
Mice
Plasmids
Poly A chemistry
Single-Strand Specific DNA and RNA Endonucleases chemistry
Transfection
beta-Galactosidase biosynthesis
beta-Galactosidase genetics
DNA, Superhelical chemistry
Deoxyribonucleases chemistry
Genetic Vectors chemistry
Nucleic Acid Conformation
Polyadenylation
Subjects
Details
- Language :
- English
- ISSN :
- 1099-498X
- Volume :
- 6
- Issue :
- 5
- Database :
- MEDLINE
- Journal :
- The journal of gene medicine
- Publication Type :
- Academic Journal
- Accession number :
- 15133767
- Full Text :
- https://doi.org/10.1002/jgm.536