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A ruler is generally used to measure the length of an object. A protractor or compass can be used to measure distance along a two dimensional curve but it is laborious. Is it possible to design a molecular ruler that can measure local bends in DNA? If so, such a ruler would be a priceless tool in several ways:
- to unravel changes in structural characteristics of macromolecules bound by proteins.
- to assist in DNA transcription regulation, recombinations, and packaging into nucleosomes.
- to provide clues to the origin of DNA replication and the recombination process.
Designing the method
To manufacture a molecular ruler, the first step is to design an experiment that will separate nucleic acid fragments according to size. How could molecules be separated by size? There are a few options:
Imagine a beaker filled with long, wiry charged molecules. Optical tweezers mimicking handheld tweezers can extract the molecules one at a time. This procedure, however, is impractical. It would take a long time to sort through even one cubic centimeter of the beaker’s contents.
Genome-sequencing robotics is another option. This process sends molecules and buffers through capillaries filled with polymers. The robots are linked to computers, which analyze the data. However, the cost of the robots and the computers, as well as the need for a laboratory specially-designed for the task, makes this procedure not easily accessible to all scientists.
- The easiest method, called gel electrophoresis, uses ordinary components similar to Jell-O to separate the molecules. To make the gel, a specific quantity of gelatin is poured into an appropriate volume of boiling water until it dissolves. Once cooled, the gel forms matrices, which have pores through which molecules can pass. The matrices needed to separate nucleic acids are usually made from polymers such as agarose or polyacrylamide.
Here is an explanation of the gel method:
- A sliver of the gel is surrounded by a buffer solution such as aqueous salt of boric acetate. A buffer is needed to maintain the acidic or basic nature of the solution, and in doing so ensures that electrical charges will remain neutral during the migration of molecules.
- The gel and the buffer are placed between a pair of electrodes and connected to a battery.
- By applying an electric charge across the gel, an electric field is generated. The force acting on the macromolecule is the product of its total charge and the external electric field that acts on it. Macromolecules such as DNA are negatively charged and they migrate through the gel towards the positive pole.
How does a negatively-charged molecule such as DNA migrate in a gel? The scientist P. de Gennes suggested that it migrates by molecular sieving.
- Longer chains move more slowly compared to shorter chains. Consider a bowl of cooked spaghetti as the gel.
- Now imagine pulling “tagged” spaghetti, the DNA, through the entangled mass with the help of tweezers.
- The DNA moves in a snake-like fashion through open spaces in the entangled spaghetti. Due to the constraints imposed by the gel matrix, these open spaces have tube-like topology.
- A new tube is formed when the leading head of the reptating chain makes favorable energetic contacts with the electric field.
Making it work
Dr. Donald Crothers’ group at Yale University utilizes the notion of molecular sieving in polyacrylamide gels to unravel the nature of DNA bending and to construct a molecular ruler. What is the basic idea? Just as scissors are an indispensable tool to a tailor, so too, is a restriction enzyme to a chemist. These enzymes cut the DNA at predetermined sites along the helical backbone.
Researchers at the Crothers laboratory have gone one step further:
- With the precision and steady hands of a brain surgeon, they extract a 6 base pair (bp) run of adenine molecules (A-tracts) of approximately 21 Angstroms in length.
- This A-tract is then inserted at the cut site of the DNA strand so that its sequence is in phase with the natural twist of the helical axis.
- The total length of the DNA is around 160 bp or about 540 Angstroms long.
What happens to the structure of the newly constructed DNA? To address this question, the DNA with the “A-tracts” and DNA without the “A-tracts” are both run simultaneously in a polyacrylamide gel experiment. Since both the DNA constructs have nearly the same length one expects similar values for its mobility. But this is not what is observed. The DNA with the “A-tracts” migrates considerably slower than the counterpart straight DNA.
The researchers learned that
- each A-tract leads to a small kink or bend of the helical axis of the DNA
- DNA with a large overall bend at its center migrates more slowly than that of the same bend located at the end of the DNA
But why is this the case? A molecule curved at the center has a smaller mean-squared end-to-end distance. This is most simply described by bending a plastic tube. A bend at the center leads to a global curvature of DNA of about 18 degrees per “A-tract.” This curved DNA molecule is in effect a molecular ruler used by Crothers to study the nature of how proteins interact with DNA.
Can a molecular ruler be designed that enables measurement of the orientation of a bend along the helical axis of DNA? Crothers group has just done this in an ingenious experiment (see Figure 1). The basic idea is similar to what happens when a stone is dropped in a placid pool of water:
Figure 1. Two intrinsic bends created by A-tracts (denoted by the light black continuous curve) in a DNA. By varying the length of the linker (denoted by the red box), a C- or S-shaped molecule can be obtained. The C-shaped construct is in cis form, while the S-shaped is in trans form.
- Emanating from the point of impact are circular waves that travel outward.
- Some of these waves interfere with each other constructively if they are in phase while others do so destructively when they are out of phase.
- If two bends are in phase, they act in unison to increase the overall bend angle.
- On the other hand if two bends are out of phase, they tend to counteract each other in such a way as to decrease the overall bend.
Since Crothers’ discovery, many scientists have asked the following question: is it possible to design molecular rulers with various geometric shapes? The phasing technique of Crothers has been used with remarkable success to create T, F, and X-shaped molecular rulers. These rulers provide a revolutionary technique for measuring local and global bends in macromolecules of biological interest. Just as with any recipe, this one for a molecular ruler requires accurate chemical ingredients, the proper reaction vessel, and a measured amount of time. Add a jolt of electricity to the gel, and voila! You’ve got a molecular ruler.
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