NRL Scientists Make Direct Measurements of Forces Between Complementary DNA Strands

Scientists at the Naval Research Laboratory (NRL) are using Atomic Force Microscopy (AFM) to measure forces between single pairs of molecules, i.e., complementary DNA strands, streptavidin-biotin, and antibodies and their antigens. "The ability to directly measure these forces with AFM and other techniques is allowing scientists to understand the physical basis of biochemical mechanisms responsible for life," says principal investigator Dr. Gil Lee, of the Surface Chemistry Branch in NRL's Chemistry Division. " NRL is focusing on using this technology to develop ultra-sensitive diagnostics to understand the molecular mechanisms responsible for biofouling, and measure the forces necessary for DNA replication. In the process, we have created a technique that can now be used by medical and scientific communities," continues Dr. Lee.

Living organisms are composed of cells that are built from macromolecules such as DNA and proteins. These macromolecules make-up both the scaffolding that holds the cells together and the motors responsible for motion. Each of these macromolecules is folded into an intricate three dimensional structure that allows it to perform its specific function. The slightest defect in a macromolecule can result in death of an organism, Dr. Lee explains.

Macromolecular structure and function are controlled by the forces between individual units that make up macromolecules and their environment. "Previously, our knowledge of these forces has been gathered from indirect x-ray crystallography and nuclear magnetic resonance measurements," says Dr. Lee. This ability to measure inter-and intramolecular forces in macromolecules such as DNA could offer unique insight into how structure produces function in these highly-important molecules. For example, the forces responsible for biological adhesion can now be directly studied at the molecular scale. Molecular adhesion controls a diverse array of phenomena such as cell migration in cancer and the adhesion of barnacles to ship surfaces.

The AFM can image surfaces both in air and under liquids at nanometer (nm) resolution. Dr. Lee notes that, " In its contact mode, the AFM lightly touches a tip at the end of a 50-to 300 micrometer-long cantilever to the sample. As a raster scan drags the tip over the sample, a detector measures the vertical deflection of the cantilever giving the local sample height. The detector typically consists of a laser reflected off the cantilever and into a position-sensitive detector. The key was to realize that if the tip and sample are coated with two types of molecules, an AFM can measure force of attraction or repulsion between them, potentially at the level of a single hydrogen bond."

NRL researchers have measured the force required to tear apart two complementary strands of DNA apart. In one experiment, according to Dr. Lee, "20-base-pair-long strands of polycytosine (i.e., single-stranded DNA) were covalently attached to the tip and DNA sample. Then, free strands of polyinosine averaging 160-base pairs long were introduced. When the tip and sample were brought together, these strands would sometimes bind to both the polycytosine on the tip and that on the sample, bridging the tip and sample. The tip and the sample were then pulled apart. The cantilever does not senseany force until the slack in the DNA is taken up, at which pointtension on the DNA begins to pull the cantilever down (starting 100nm of separation). The form of the force-distance curve describes the Figure 2 mechanical properties (intramolecular forces) of a single strand
of DNA as it is stretched from a random coil into a linear molecule.
When the force is large enough (-600pN), the intermolecular DNA-DNA
bonds at either the tip or sample break, and force on the cantilever returns to zero."

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Figure 1. Concept of AFM and the optical lever; (left) the optical lever; (right) close-up of the cantilever touching the sample. The sample, attached to the piezoceramic translator, moves underneath the cantilever. Scale drawing: The piezoceramic measures 24mm in diameter, while the cantilever is 100 micrometers long (Figure courtesy of D. B. Baselt).

Figure 2. Interaction force between two complementary strands of DNA, measured by AFM. Relative surface displacement is the distance between the tip and sample relative to the position at which 1,000pN of force is reached. Measurements are recorded both as the tip and sample are brought together (thin trace) and as they are separated (thick trace).

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