In Vivo DNA Stabilizer for Nuclease Resistant DNA
VivoSyn® enables the in vivo production of nuclease resistant DNA or RNA phage, DNA or RNA viruses, genomic DNA, etc. Cells are grown in media using the phosphate analogue in place of phosphate resulting in nucleic acids that are completely modified. Such chemically modified DNA or RNA may be useful for DNA vaccine adjuvants, gene therapy, and/or the isolation of these and related nucleic acids from cells. Below are examples showing the resistance of genomic DNA (Fig. 1) and single stranded phage DNA (Fig. 2) to DNAse 1 digestion as well as assay for extent of modification (Fig. 3). Efficiency of incorporation into plasmid DNA is greater than 90% when using media fully substituted with the analogue. Note plasmid modification of purified DNA is not stable in vitro for more than two days. However, the modification is stable in single-stranded DNA or RNA.
1. Digestion of Goldfish DNA
2. Digestion of M13 ss Phage DNA
Goldfish were maintained in an aquarium with or without the phosphate analogue. Lanes 1-4 show wild type DNA and Lanes 5-8 resistant DNA treated with DNase I for 0, 5, 10, and 15 min respectively. Note that some digested DNA at the bottom of Lns 4-6 is due to the fact that not all the cells in the intestinal gut preparation where actively dividing and able to readily incorporate the analogue.
Lanes C and P are control lanes without DNase I for normal and phosphate substituted DNA. The two forms of phage DNA indicated by arrows: closed circular (I) and linear (II). Conversion of form I to II during DNase 1 digestion was assayed at 1, 2, and 3 hrs. (lanes 1-3 and 4-5 respectively). By two hrs. (lane 2) form I is completely digested in normal phage DNA samples. In contrast, using DNA from cells grown in phosphate substituted media, no conversion is detected for up to 3 hrs. of incubation (lanes 4-6).