Lowering Salt Content in DNA Solutions May Improve Gene Therapy Success
Scientists are seeking to understand the natural mechanism of DNA condensation into nanostructures -- in particular, toroids, which look like tightly wound garden hoses. Densely packed DNA is nature's efficient way of transporting genetic information, done particularly well by sperm cells and viruses.
Researchers want to mimic this process to improve DNA delivery for gene therapy and DNA-based vaccines, but they face many challenges in the laboratory where DNA in solution typically exists in an extended, rather than condensed state. Scientists have been able to cause DNA to condense into toroids by adding positively charged molecules to samples, but they have had difficulty finding the right molecules to achieve consistent, optimal toroid sizes of less than 50 nanometers.
However, scientists at Georgia Institute of Technology have made a significant advance in controlling the size of DNA toroids. In the July 18, 2003 online issue of the journal Proceedings of the National Academy of Sciences (PNAS), they report that reducing salt concentrations below normal laboratory solution levels shrinks both the diameter and thickness of DNA toroids. This finding resulted from a combined investigation of how static DNA loops and solution conditions might be used to control toroid dimensions.
"But even without static loops present, DNA produces smaller toroids if you reduce the salt concentration," said Nicholas Hud, an associate professor of biochemistry who is leading the study funded by the National Institutes of Health. "We found a systematic relationship between reducing salt and reducing toroid size. It is surprising that such a study was not previously done because salt concentration is such a fundamental parameter in studying molecules in solution, particularly such highly charged molecules as DNA."
Protocols for preparing DNA for delivery to cells often call for salt conditions that differ from those DNA encounters when injected into body tissues, Hud noted. "If you change the salt conditions during DNA delivery, it will change particle size and have a dramatic effect on the efficiency of gene delivery," he added. "This could explain why some researchers aren't getting as good a rate of transfection (the incorporation of DNA into a cell) as they should."
In the study reported in PNAS, Hud and his Ph.D. students Christine Conwell and Igor Vilfan also describe using the positively charged, inorganic molecule hexammine cobalt (III) to condense a DNA molecule containing a specially designed sequence. The synthetic sequence causes a region of the DNA molecule to bend into two loops of 25 nanometers each in diameter. In other words, these nanoscale loops were "programmed" into the DNA sequence.