Rahul Rattan1, Anna U Bielinska, Mark M Banaszak Holl. 1. 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.
Abstract
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.
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.
Authors: D Lechardeur; K J Sohn; M Haardt; P B Joshi; M Monck; R W Graham; B Beatty; J Squire; H O'Brodovich; G L Lukacs Journal: Gene Ther Date: 1999-04 Impact factor: 5.250
Authors: Seungpyo Hong; Rahul Rattan; István J Majoros; Douglas G Mullen; Jennifer L Peters; Xiangyang Shi; Anna U Bielinska; Luz Blanco; Bradford G Orr; James R Baker; Mark M Banaszak Holl Journal: Bioconjug Chem Date: 2009-07-07 Impact factor: 4.774