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.
J Gene Med. 2014 Mar-Apr;16(3-4):75-83. doi: 10.1002/jgm.2761.
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 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).
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.
尽管细胞质 DNA 降解在降低转基因表达方面起着重要作用，但质粒降解模式在很大程度上仍未得到探索。
采用 Illumina 染料测序技术，为 S1 核酸酶和细胞质核酸酶提供降解位点信息。S1 核酸酶作为琼脂糖凝胶法和测序方法比较的阳性对照。
β-内酰胺酶基因和巨细胞病毒（CMV）启动子之间的多聚（A）区域被确定为多聚体处理细胞质中最有可能的切割位点。通过凝胶电泳和测序确定了第二个最可能的位点，即在β-内酰胺酶基因的 5'端。在 OriC 区域、SV40/多聚（A）区域、荧光素酶基因和 CMV 启动子中检测到其他位点。用对照细胞的细胞质处理的质粒的序列分析显示，OriC 区域、β-内酰胺酶基因和紧随荧光素酶基因的多聚（A）区域的切割活性最大。其他切割活性区域包括 SV40 启动子和β-内酰胺酶多聚（A）终止序列。细胞质核酸酶和 S1 核酸酶在细菌复制起点（OriC）都表现出很强的活性。
高通量质粒测序揭示了荧光素酶质粒 DNA 序列中对细胞质核酸酶降解敏感的区域。这为改进质粒和/或聚合物设计以优化蛋白表达的可能性提供了新的靶点。
