The role of polyadenylation signal secondary structures on the resistance of plasmid vectors to nucleases.
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.


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.


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.
Ribeiro
Sofia C
SC
Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal.
Monteiro
Gabriel A
GA
Prazeres
Duarte M F
DM
D000818:Animals; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D003412:Cricetulus; D004278:DNA, Superhelical / Q000737:chemistry*; D003851:Deoxyribonucleases / Q000737:chemistry*; D005822:Genetic Vectors / Q000737:chemistry*; D013006:Growth Hormone / Q000737:chemistry; D051379:Mice; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011061:Poly A / Q000737:chemistry; D026723:Polyadenylation; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004278:DNA, Superhelical; D011061:Poly A; D013006:Growth Hormone; D003851:Deoxyribonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D001616:beta-Galactosidase