Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.
BACKGROUND
Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored.


METHODS
Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches.


RESULTS
The poly(A) region between the β-lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex-treated cytosol. The second most likely site, at the 5' end of the β-lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the β-lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the β-lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication (OriC).


CONCLUSIONS
High-throughput plasmid sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, MI, USA.
Rahul
R
Rattan
Anna U
AU
Bielinska
Mark M
MM
Banaszak Holl
D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics; D010957:Plasmids / Q000378:metabolism*; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D019076:Transgenes; D001618:beta-Lactamases / Q000235:genetics
C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D039102:Poly(A)-Binding Proteins; D004247:DNA; D008156:Luciferases; D004720:Endonucleases; D001618:beta-Lactamases
HeLa; biotechnology; plasmid design; plasmid gene delivery; polymer vector