Researchers in the UW department of chemistry have developed powerful probes of molecular structure using the technique of nuclear magnetic resonance spectrometry (NMR) to tackle, among other tasks, the problem of determining the structure of large, complex biomolecules like DNA. Moreover, UW faculty have teamed up with scientists at Pacific Northwest National Laboratory (PNNL) in Richland, Washington, to design and construct the world's most powerful NMR instrument.
The NMR technique helps scientists deduce the arrangement of atoms within a molecule--its molecular structure. Molecules consist of atoms linked together by chemical bonds. Atoms, in turn, consist of a dense, compact core, called the nucleus, surrounded by what could be likened to a "cloud" of electrons. The electrons participate in chemical bonding, providing the chemical "glue" that holds atoms together to make a molecule. The exact arrangement of the atoms determines the identity of the molecule, its structure, and its chemical characteristics. But since atoms and molecules are too small to see, chemists have devised a number of complementary strategies to deduce how atoms are linked together.
One such strategy is NMR. The method is based on measuring the absorption of very-low-energy radio waves by the nuclei of atoms in a molecule which has been placed in a magnetic field. The particular distribution of electrons around a nucleus influences the precise frequency of the radio waves that are absorbed. That electronic distribution in turn is a function of the chemical bonds and neighboring atoms surrounding the nucleus. So, by measuring the frequencies at which atoms absorb energy, scientists may gain clues about the local environment of each atom and can then deduce the overall structure of the molecule. In practice, several different but related NMR procedures are often used in concert to piece together a picture of a complex structure. An extension of nuclear magnetic resonance spectrometry to the imaging of biological tissues is magnetic resonance imaging, or "MRI."
In about 1980, the UW chemistry department under the direction of then-chairman Alvin L. Kwiram began an initiative to build up its program of NMR research. Kwiram recruited Brian Reid, and subsequently Gary Drobny, to the chemistry faculty to launch a new era in NMR research. Reid, a biochemist, began working on the structure of DNA in solution, using sophisticated, high-resolution NMR techniques devised and refined by Drobny, a physical chemist.
Prior to this work, the only way to determine the structure of such a molecule was to grow a solid crystal of it, and then analyze it by x-ray crystallography; but crystallizing the substance, even if possible, can cause structural distortions compared to the configuration of the molecule in its natural state in biological fluids.
Reid and colleagues pioneered a groundbreaking approach to solve the structure of DNA in solution by measuring literally hundreds of inter-nuclear distances using NMR and then developing a computer algorithm to deduce the molecular arrangement of the atoms. Thus, for the first time, a revolutionary new method was available for determining the structure of large molecules in solution to complement the century-old traditional method of x-ray crystallography with crystalline materials. These NMR methods are now among the most powerful tools used by chemists and life scientists to study the solution structure of biomolecules.
More recently, Reid has used a similar procedure to determine the structure of hybrid double helices--biomolecules similar to double stranded, helical DNA but containing one strand of ribonucleic acid (RNA) and one strand of deoxyribonucleic acid (DNA). "These hybrids are very important in such processes as the life cycle of retroviruses like HIV [Human Immunodeficiency Virus]," the cause of AIDS, explains Reid.
Reid and colleagues have established for the first time the unique structure of the hybrid double helix. "It is distinctly different from the DNA double helix or RNA double helix," says Reid. "It's the uniqueness of the hybrid that explains how the enzyme ribonuclease-H works--an enzyme that is very important in the HIV replication cycle, and on which an awful lot of research on AIDS is being focused."
In addition to the hybrid double helix work, Reid and his students have been using NMR to study abnormal base pairing in DNA. Although Watson and Crick established that in the DNA double helix, the base adenine (abbreviated "A") pairs with thymine (T), and guanine (G) pairs with cytosine (C), Reid's group has established that, in certain sequences, G can pair with A and vice versa. In one of these sequences, which is repeated thousands of times and makes up 5-6% of human DNA, the DNA strand pairs with itself instead of with its complementary strand, using GA pairing instead of GC pairing to form what Reid calls "sticky DNA." He feels that this stickiness may explain how duplicated chromosomes are segregated equally into daughter cells at mitosis. "Although the process is poorly understood at the molecular level, the accurate segregation of duplicated chromosomes during cell division is the basis underlying all of genetics and evolution" says Reid.
An unexpected spin-off from Reid's exploration of GA base pairing in DNA is already shedding some light on a newly discovered class of inherited genetic diseases known as triplet-repeat diseases. These include Huntington's disease and a variety of neuromuscular dystrophies and atrophies; recently they have all been shown to involve the expansion of a string of adjacent GCA triplets in the corresponding target genes (e.g., GCA GCA GCA GCA GCA, etc.). The expansions range from 10-20 repeats in the normal population to hundreds of repeats in those with the disease.
These diseases all show the phenomenon of anticipation—that is, increasingly longer repeats within the affected gene from one generation to the next, resulting in increasingly severe symptoms and earlier age of onset. Also, the diseases exhibit autosomal dominance, in which only one of the two chromosomes need be affected to cause the disease. "It's chemically fascinating that in all these diseases it is always the GCA word (out of the 64 words in the genetic dictionary) that expands," says Reid. He suspects that his unusual GA pairing is involved in the expansion process.
Meanwhile, UW chemistry professor Gary Drobny and colleagues have been working on the design and construction of the world's first 1-Gigahertz NMR spectrometer in collaboration with Paul D. Ellis of the Environmental and Molecular Sciences Laboratory (EMSL) at PNNL in Richland, Washington, operated by Battelle for the U. S. Department of Energy. The 1-Gigahertz spectrometer will be one of the centerpieces in an array of advanced instruments and computing tools for the study of environmental systems at the new EMSL facility.
One Gigahertz (GHz), equivalent to 1,000 Megahertz (MHz) or 10 Hertz (cycles per second), is an indirect measure of the strength of the magnet used in the instrument. (It reflects the resonance frequency for protons in that field strength). The new magnet, which is being constructed for the project by Oxford Instruments in the U.K., will be one of the largest magnets of its type ever made, says Drobny. It will operate at a temperature of 1.8 Kelvin and will employ special superconducting alloys such as niobium-tin and niobium-titanium in order to achieve its powerful field.
The quest for ever higher field strength is important because in large molecules, NMR lines overlap, which limits the ability to resolve them and obtain structural information. But the amount of overlap decreases as the strength of the magnetic field increases. Until this project was launched, most researchers had access, at best, to instruments with field strengths of 500 MHz or lower.
The new apparatus represents a significant increase in resolving power, permitting the study of larger molecules than ever before, as well as of solids, especially biomaterials and catalysts. The spectrometer will be available part of the time for use by scientists elsewhere, serving as a "prime resource" for efforts nationwide in environmental and biological sciences, says Ellis.
It should be noted that none of these developments in super-high field NMR would have been possible in the Pacific Northwest without the strong support of the Murdock Charitable Trust.