How DNA topology and DNA length affect the body's defense against nucleic acids of invading organisms in the blood
Seminar Room 1, Newton Institute
It has long been known that human blood contains enzymes that digest DNA to protect the body against invasion by foreign organisms. We set out to determine how DNA length and supercoiling affected DNA vector survival in human serum. Closed circular, supercoiled vectors ranging from ~300 to ~4,000 bp were incubated at 37°C in human serum. Aliquots were taken over several days and were analyzed by gel electrophoresis. We found that digestion in human serum strongly correlated with increasing DNA length. To our surprise, we also uncovered a trend by which serum proteins bound and protected DNA. We recently published that the compaction by DNA supercoiling protected small (<1,200 bp) DNA circles from the mechanical shear forces of aerosolization or sonication (Catanese et al. 2012); we hypothesized that a similar trend would be observed with human serum degradation. This hypothesis proved incorrect because linear and nicked DNA survived ~ 3- to 6-fold longer than supercoiled DNA. These results agree with previously published data showing that DNAse I, the major nuclease in human blood, preferentially acts on supercoiled DNA. Together, these data support a model in which foreign DNA is gapped by nucleases in the bloodstream and that this action is enough to hamper replication and transcription of DNA vectors in human cells. In addition to explaining how the blood protects humans from invasions, this understanding opens the door for designing DNA that is relaxed, closed circular, and tiny for gene therapy to be delivered intravenously. The lack of supercoiling allows the circles to persist longer and reach their target cells where supercoiling will then be restored, resulting in transcription and gene expression.
This work was supported by NIH RO1AI054830, Human Frontier Science Program, and Seattle's Children's Hospital Research Foundation, part of NGEC, to L.Z. T.J.B. was supported by NIH Grant T32 GM88129.