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Cooking Up a Molecular Ruler

Jayanta Fowler Mohanty


Molecular rulers can measure:

  • bends in DNA
  • how proteins interact with DNA
  • nano-scale structures
  • growth of genetic structures

September 2000

Note: Because some of the information in this article may be outdated, it has been archived.

Will molecular rulers measure nano-scale structures such as those found in DNA?

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

Is is possible to separate different-size molecules?

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.

Inexpensive gel can be used to design a molecular ruler.
  • 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:

Molecules are zapped by electricity to sort them.
  • 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.

The process is similar to molecules falling through a sieve.
  • 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

Enzymes can be used to cut portions of DNA.

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

DNA molecules have bends which can function as a molecular ruler.
  • 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.

What’s next?

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.

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.

Molecular rulers can measure the degree of DNA bend.
Conclusion: Differently shaped molecular rulers will measure different macromolecular structures in the near future.
  • 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.

Jayanta Fowler Mohanty is a senior at Cumberland High School in Cumberland, Rhode Island. He credits his interest in the polyelectrolyte behavior of DNA to the work of Nobel Laureate Steven Chu of Stanford University. Chemical Physical Letters recently published Jayanta’s article “Anomalous migration of DNA.” In May 2000, Jayanta won the First Prize - Grand Award for Chemistry at the International Intel Science and Engineering Fair.

Cooking Up a Molecular Ruler

Scientists create molecular ruler

2001-02-14 News article, “Scientists Create Molecular Rulers Enabling Precise Construction of Nanoscale Structures,” about a breakthrough in the development of a molecular ruler, enabling the construction of nanoscale structures.

Picture gallery of DNA

In this gallery, images show the kinks, or bends, in DNA.

Steven Chu bio

This Nobel laureate inspired the author to pursue research on the molecular ruler.

The Crothers lab

This lab made breakthroughs in the design of molecular rulers.

Call for math and science partnerships

The National Science Foundation is seeking government funding for states and local school districts to work with mathematicians, scientists, and engineers of higher education in order to help raise math and science standards and provide math and science training for teachers in K-12 education. The NSF is requesting comments on this initiative. Read their statement and, if you agree, also write a letter to your congressman in support.

For teachers: biochemistry resources

Project Galileo offers ready-to-use materials to teachers of chemistry and biochemistry. Registration and login are required.

Genomics Analogy Model for Educators (GAME)

The GAME website is a tool for high school science teachers and higher education instructors who teach genomics but who do not have a molecular biology background. Useful analogies and resources are available for teachers to use in their classroom.


Biointeractive is a website and a collection of biology-focused teaching materials created by the Howard Hughes Medical Institute. Many materials are available to educators for free and can be ordered from the catalog. The site’s information and links are also useful to high school seniors and college-level students.

  • »Haran, T.E., Kahn, J.D., Crothers, D. M. J. Mol. Biol, 1994, 244:135-143.
  • »Kahn, J.D., Yun, E., Crothers, D. M. Nature 1994, 368:163-166.
  • »Kerppola, T. K. Proc. Nat. Acad. USA 1996, 93:10117-10122.
  • »Drak, J., Crothers, D. M. Proc. Nat. Acad. 1991, USA 88:3074-3078.
  • »Perkins, T.T., Smith, D.E., Chu, S. Science 1994, 264:819-822.


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