For Immediate Release
Researchers have found they can control the size of densely packed DNA
structures by changing the salt concentration in solutions containing
DNA. The finding could improve the efficiency of gene delivery for medical
treatment and disease prevention.
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.
Hud theorized years ago that the spontaneous formation of loops along DNA is the first step necessary for toroid formation, and a key factor in determining toroid size, he said.
"Now, we've made these loops always present there," Hud explained.
"When we add positively charged molecules that bind to the DNA, the
loops provide a built-in starting point for DNA condensation. The loops
also act as a template upon which the rest of the DNA rolls up to form
a toroid. The toroid forms because the positively charged molecules make
DNA want to stick to itself
. and we found that our static loops
reduce DNA particle size and tighten particle distribution."
Hud describes as serendipitous the additional finding that lowering the
salt concentration in DNA solution also reduces the size of toroids. Together,
these results helped Hud's team develop models for DNA toroid formation.
The researchers' data can now serve as a test for theoretical models of
DNA condensation, they say.
Meanwhile, Hud's team is exploring how the order in which they add salts
to DNA solutions affects particle size and shape -- whether salts should
be added before or after the DNA condensation process is prompted by positively
"By studying the fundamental process of DNA condensation we hope
to determine all the factors that help produce particles of smaller size
and narrower size distribution. The combined effects of these factors
should help us to produce the optimal particles for gene delivery,"
He believes a systematic approach to the high-stakes goal of developing
an efficient, artificial gene delivery method will pay off.
"There have been a lot of attempts to improve DNA delivery by simply mixing molecules and empirically testing to determine their efficiency," Hud said. "But DNA condensates are difficult to understand . We might be missing something in our information about what's happening from the lab bench to the delivery of DNA to cells. We want to understand the nature of DNA particles all the way from the test tube to the cell."
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