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Even Molecules Act Like Individuals, Stanford Researchers Discover

Date:
February 19, 1998
Source:
Stanford University
Summary:
Molecules, it seems, can exhibit a surprising degree of individuality. In experiments that examine the physical behavior of single molecules, Stanford researchers have discovered that when identical polymers - long, flexible, spaghetti-like molecules that are found in everything from plastics to living cells - are prepared in the same way and exposed to the same conditions, they unfold in a variety of different ways.

Molecules, it seems, can exhibit a surprising degree of individuality. In experiments that examine the physical behavior of single molecules, Stanford researchers have discovered that when identical polymers - long, flexible, spaghetti-like molecules that are found in everything from plastics to living cells - are prepared in the same way and exposed to the same conditions, they unfold in a variety of different ways.

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"Molecular individualism is a new surprise. We discovered it because we have developed the ability to visualize and manipulate single molecules. This ability is allowing us to address a number of long-standing questions in polymer science," said Nobel laureate Steven Chu, the Theodore and Frances Geballe Professor of Physics and Applied Physics at Stanford. He spoke on Friday, Feb. 13, at the annual meeting of the American Association for the Advancement of Science in Philadelphia.

Limitations of bulk studies

Previously, scientists have been limited to studying polymers in bulk, by the millions and billions. Chu likens it to trying to determine the nature of animals in a zoo using only information about averages. "If someone did a series of experiments that only measured the average size, weight and number of legs of the animals, he would get a distorted picture. For example, he might find that the average number of legs on the animals is 2.7, and then look for a theory of animal development that could explain his finding. Only by looking at individual animals can you get a true sense of the diversity of species," Chu said.

Beginning in 1989, Chu's laboratory has developed techniques that allow them to study the behavior of individual polymers using readily available strands of viral DNA. Although these molecules are normally too small to view with an optical microscope, the researchers have made them visible by attaching a string of fluorescent dye molecules. This has allowed Chu - working with doctoral students Thomas T. Perkins, now a postdoctoral student at Princeton University, and Douglas E. Smith - to discover that 20 years of experimental evidence regarding the way that polymers unfold did not reveal that identical molecules exposed to the same conditions might act very differently.

In these experiments, Chu and his students observe how floating strands of DNA unravel when exposed to microscopic currents. Such currents, or flow fields, occur as a fluid passes through any constriction or nozzle. How polymers deform in these fields is directly related to how they behave during commercial processes like injection molding.

A microscopic flow cell

The researchers used microfabrication techniques to etch two flow channels in the form of a cross. The channels are about 25 human hair widths (650 microns) wide and about 10 hair widths (220 microns) deep. By simultaneously pumping liquid into the ends of one channel and sucking it out of the ends of the other channel, the scientists create microcurrents at the very center of the cross that pull outward along one axis and push inward along the other.

In their first procedure, they allowed the randomly coiled DNA molecules to flow down one channel until they reached the center of the flow cell. When the molecules reach the centerpoint, the microcurrents force them to unravel. Despite taking great care to use identical strands of DNA in identical flow conditions, the experimenters were surprised to observe the diverse ways that they unraveled. Some remained stubbornly coiled. Others unraveled from a kink in the middle. Others formed intermediate dumbbell shapes, with knots at one or both ends. Most surprisingly, some got caught in a folded shape that took much longer to unravel completely.

Their initial results were published in June 1997 in the journal Science. Since then the researchers have improved and extended their experiments. Fifteen years of data explained

The single-molecule studies showed that polymer scientists have been misinterpreting the results of some bulk experiments for the last 20 years. During this time, the scientists inferred the extension of the polymers by measuring how the polarization of a laser beam changes when it passes through flowing polymer mixtures. The polarization was seen to plateau to a constant value as the flow gradient increased; this led to the inference that the molecules had become fully extended. Other polymers were also observed to fracture at their mid-points, also suggesting that they were fully extended. In the last several years, however, contradictory evidence based on light scattering experiments raised suspicions about this interpretation.

"We decided to have a new look at this problem," Chu said. By observing the behavior of individual DNA molecules directly with an optical microscope, he and his students have shown that, at the point where scientists had thought the polymers should all be fully stretched out, a significant proportion of the molecules remain partially coiled. By measuring the length of those molecules that have reached their equilibrium state, they have decisively verified a long-standing prediction by a leading theoretician in the area, Nobel laureate Pierre-Gilles de Gennes of the Coll?ge de France.

However, the experiments in the Chu lab revealed a surprising discovery: The molecules looked different as they unraveled. Furthermore, the rate at which they unraveled depended on the way they looked. The fact that polymers might unravel in radically different ways was not considered by theorists and experimentalists prior to this observation. In a "commentary" accompanying the Science article, de Gennes noted the significance of what he called "molecular individualism.2 However, he suggested that the individualism may be a genuine new effect or it may be due to a slight pre-deformation before the polymers entered the field of view. Although Chu and his colleagues took special precautions in the original work to insure that this was not the case, Chu and Smith decided to improve the experiment by suddenly turning on the fluid flow so that they could watch the polymer unravel beginning from the initial randomly coiled state.

When Smith duplicated the conditions of the first experiment, he got exactly the same results. So he began subjecting the molecules to much higher stretch rates, and the proportion of the intermediate shapes changed dramatically. The percentage of dumbbell and coiled shapes decreased and the kinked and folded shapes became dominant.

"The data have proved to be extremely rich and can be analyzed in many ways," Chu said. His lab has been working in collaboration with Ron Larson at the University of Michigan, who has developed computer simulations of the behavior of the polymers. This work strongly suggests that the "molecular individualism" arises from exceedingly small differences in the initial configuration of the polymer.

"We have found that random thermal fluctuations in the initial starting point of the elongation get magnified into dramatic differences," Chu said. For instance, simply rotating a coiled polymer with respect to the velocity flow will completely alter the way that it unravels.

"We have shown that very different behavior can be seen with identical molecules in the same environment. The unraveling is due to tiny differences resulting from thermal fluctuations in the starting conditions," Chu said.

Chu emphasizes that the process involved is totally different from popular chaos theory, in which very small differences in initial conditions can have large and unpredictable effects on the course that a given system will take. In chaos theory, the unpredictable results arise from the nonlinear character of the equations of motion. In the case of molecular individualism, the equations that govern the behavior can be linear.

Nevertheless, this new phenomenon similarly dramatizes the role that random factors play in a wide range of physical and biological processes, Chu said. "It should occur in systems where equilibrium statistical mechanics does not apply. Equilibrium statistical mechanics assumes that the behavior of a collection of molecules can be predicted by considering all the states of the system accessible at some finite temperature. However, if we examine an individual molecule and force it to do something on a time scale faster than its natural relaxation time, the molecule may be forced into states that are much different from those predicted by thermal equilibrium," he said.

That is what happens in the case of the polymer unfolding. There are two different time scales involved. One is the time it takes the polymer to react, called the relaxation rate. The other is the stretch rate exerted by the liquid. When the stretch rate is faster than the relaxation rate, then molecules can get caught in a folded configuration that is normally unstable.

"It's a case of things happening so fast that the right hand [of the molecule] doesn't know what the left hand is doing," Chu said. "Such a situation can also arise in the case of protein folding. It has been conjectured by theorists that there may be several distinct intermediate (partially folded) states on the way towards the final 3-dimensional structure. This behavior has yet to be seen experimentally."

Speaking tongue-in-cheek, the Nobel laureate noted that the accidental factors play a greater role in everyday life than most people realize or accept. "In our contribution to the 'nature versus nurture' or 'genes versus environment' debate, we put identical DNA (DNA strands that we work with are all from the same lambda phage virus) in the same environment and say 'Go!' Our subjects begin to look different and some of the individuals get to where they are going much faster than others. [Before this] we did not consider how important tiny random influences would be," Chu said. "Perhaps such accidental influences play an important role in how our lives evolve as well. If you could clone Einstein 100 times, would you get 90 Einsteins? Would you get one?"


Story Source:

The above story is based on materials provided by Stanford University. Note: Materials may be edited for content and length.


Cite This Page:

Stanford University. "Even Molecules Act Like Individuals, Stanford Researchers Discover." ScienceDaily. ScienceDaily, 19 February 1998. <www.sciencedaily.com/releases/1998/02/980219061932.htm>.
Stanford University. (1998, February 19). Even Molecules Act Like Individuals, Stanford Researchers Discover. ScienceDaily. Retrieved November 26, 2014 from www.sciencedaily.com/releases/1998/02/980219061932.htm
Stanford University. "Even Molecules Act Like Individuals, Stanford Researchers Discover." ScienceDaily. www.sciencedaily.com/releases/1998/02/980219061932.htm (accessed November 26, 2014).

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