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Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.

ABSTRACT: Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored.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.The poly(A) region between the ?-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 ?-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 ?-lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the ?-lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication (OriC).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.

SUBMITTER: Rattan R

PROVIDER: S-EPMC4071455 | biostudies-literature | 2014 Mar-Apr

REPOSITORIES: biostudies-literature

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Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.

Rattan Rahul R Bielinska Anna U AU Banaszak Holl Mark M MM

The journal of gene medicine 20140301 3-4

<h4>Background</h4>Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored.<h4>Methods</h4>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.<h4>Results</h4>The poly(A) region between the β-lactamase gene and the cytomegalovirus ...[more]

PMID: 24700644

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Project description:In this work, we used our recently developed method Spec-seq to characterize the binding specificity of Glucocorticoid receptor in vitro. This GR Spec-seq experiment has been run twice separately (Dec 2014 and Mar 2015) with different library compositions. The basic workflow is the same as our previous work for lac repressor published in Genetics 198.3 (2014): 1329-1343. Recombinant human GR protein was used to facilitate in vitro DNA-binding and separation experiments. Bound and Unbound DNA fragments were separated in EMSA gels, purified, barcoded for further Illumina sequencing. An initial experiment was performed using the putative consensus sequence: AGAACA GGG TGTTCT; randomized library 1: AGAACN NSN NGTTCT [diversity =512]; randomized library 2: AGAANN GGG NNNTCT [diversity = 1024]; randomized library 3: AGAACA GGG TGNNNN [diversity =256]; randomized library 4: AGAACA GGGC NNNTCT [diversity = 64]. The initial total library diversity was ~1853 with a library composition of 10% positive control sequence + randomized libraries 1-4 + 5% negative control sequence. A 5th library containing 2304 sequences based on DDNACW KKN KGTTCT, where D="not C", N="any base", W="A or T" and K="G or T" was subsequently prepared and analyzed using similar ratios of control sequences. Binding conditions for EMSA were 100ng FAM-labelled dsDNA+ 0/0.5/1/2/4uM GR DBD protein for each lane, 1X NEB buffer 4. GR DBD was prepared as previously described. The EMSA was performed using a 9% 33:1 acrylamide gel and TB buffer, and was run at 200V for 30mins @ 0 degrees C. The 2uM protein lane used for final sequencing. Bound/unbound fractions resulting from EMSA of these libraries and conditions were used to generate PWMs as described. The GR PWM that was generated through this analysis was used to define relative binding energies using the patser program (35), which can be accessed online at (http://stormo.wustl.edu/consensus/cgi-bin/Server/Interface/patser.cgi ). Derived binding affinities are proportional to the inverse of the natural log of the calculated energies.

Dataset Information

Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.

Publications

Quantification of cytosolic plasmid DNA degradation using high-throughput sequencing: implications for gene delivery.

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