We are developing new methods for single-molecule DNA sequencing. We are interested in how genes work, and would like to study the way in which proteins change DNA structure to switch genes on and off. We are also interested in the chemistry and physics of the liquid-solid interface, and are trying to understand electrochemical and charge transfer processes at the single-molecule level. To carry out our experiments, we work at building specialized scanning probe microscopes in collaboration with our industry partner, Molecular Imaging Corporation (Now Agilent).
Key research project ongoing within our center include:
Electronic Sequencing by Recognition
DNA testing is transforming health care and medicine, but current technologies only give a snapshot of an individual’s genetic makeup. Any patient wanting a complete picture of their inherited DNA, or genome, would drop their jaw at the sight of the bill - to the current tune of a million or more charged for every human or mammalian-sized genome sequenced.
Now, our center's scientists are focused on expanding efforts to dramatically lower the cost of DNA sequencing.
The NHGRI, part of the National Institutes of Health (NIH), has set an ambitious target of $1,000 or less - a cost 10,000 times lower than current technology - to make genome sequencing a routine diagnostic tool in medical care. The reduced cost may allow doctors to tailor medical treatments to an individual’s genetic profile for diagnosing, treating, and ultimately preventing many common diseases such as cancer, heart disease, diabetes and obesity.
Our ambitious DNA sequencing research combines physics, chemistry and nanotechnology with engineering. Much like the computer industry, DNA sequencing technology is driven by the mantra of faster, cheaper and more reliable. In the past generation, sequencing costs have fallen 100-fold, from roughly a dollar a DNA base to a penny, but are still far out of reach for the public.
Our researchers have been charged with the daunting task of shrinking down the 13-year, $2.7 billion Human Genome Project to mere days. Our vision would enable scientists to sequence billions of base pairs of DNA in a single day. This is the size of an average mammalian genome and is approximately 10,000 times more bases per day than can be sequenced using current technologies. Only by increasing the speed of sequencing and reducing its cost, can genetic research develop a more significant role in everyday medical practice.
One way that single-base resolution might be achieved in a nanopore reader is to use electron tunneling to sense the bases. Electron tunneling is a localized effect. Vacuum tunneling rates decrease by about a factor of ten for each Angstrom that a gap between metal electrodes is increased. Thus, a system based on electron tunneling should only sense the base in the immediate vicinity of the electrode. Motivated by this fact, Zwolak and DiVentra1 have proposed that two electrodes, placed very close to a nanopore, could sense the chemical nature of a base via the magnitude of the tunnel current through each base as it passed between the electrodes. Simulations suggest that, with a carefully chosen bias, such discrimination may indeed be possible. The scheme is difficult to implement. The electrodes must be placed within an atomic diameter or so of the target sites on the DNA, the DNA must be oriented, and fluctuations suppressed. While it might be possible to constrain and align the DNA using a large electric field, other factors, such as contamination on the electrodes,2 and the random presence of water molecules and ions all pose problems. These are the very problems that have plagued molecular electronics for years. For instance, DNA has been described as an insulator,3 a semiconductor,4 a metal5 and even a superconductor.6 The most likely source of this extreme range of results lies in experimental methods, problems that now appear to be largely removed.7 The key to making reliable electronic measurements on single molecules lies in using chemistry to form predictable bonds between the metal electrodes and the molecule.8, 9
The bonds used to contact the molecules to the metal do not have to be strong and covalent. Ohshiro and Umezawa showed that tunnel current between bases could be significantly enhanced by hydrogen bonds.10
Can this scheme generate single-molecule base-specific information? A simple model of the base-base interaction can be built by attaching one base to the probe of a scanning tunneling microscope (STM) and another (in the form of a thiolated nucleoside) to a conducting substrate. The movement of DNA past the "reading head" can be simulated by pulling the STM probe away from the surface and recording the decay of tunnel current. The results are quite striking11 and some of them are summarized in figure X(B - D). Figure X(B) shows data for a bare metal surface with various functional groups on the probe, X(C) data for a surface functionalized with thymidine and X(D) data for a surface functionalized with deoxycytidine. The green and orange curves are for a bare probe and a thiophenol-functionalized probe. The black curves are for a guanine functionalized probe. Notably, signals obtained with the control group are insensitive to the chemical nature of the group attached to the tip. This illustrates the role played by the "weak link" (the non-bonded interaction at the bottom surface). A 100 MW resistor in series with a 100 kW resistor is still basically a 100 MW resistor. The signals obtained over a thymidine coated surface (figure X(C)) are notably different. The G-T mismatch is stabilized by two hydrogen bonds and the signals obtained with a guanine on the probe are clearly larger than the controls. The really striking difference occurs for the Watson-Crick G-C basepair (figure X(D)). The signal is considerably larger. Analysis of these signals suggests that good discrimination is possible and that a small number of repeated reads could lead to highly accurate sequence data.11 A similar study of the entire "sandwich" (electrode-guanidinium-DNA-base-electrode) suggests that the readout will work on intact and unmodified single-stranded DNA.12
Should it prove possible to implement a similar scheme in conjunction with a nanopore, this readout scheme could offer some advantages. The junction is self-assembling, driven by the hydrogen bonding. Thus manufacturing and assembly tolerances are reduced relative to the direct tunneling scheme. The readout is chemical with a "binary" detection. A junction either forms or it does not. In practice, mismatches also give signals, but these appear to be quite distinct from the matched reads. It is even possible that the transient hydrogen bonding could provide the much needed "clock" to control the translocation rate through the pore. A disadvantage of the scheme is that, in the embodiment shown here, one reader is required for each base. Thus alignment would get complicated unless a homogeneous set of DNA fragments was presented to the reader.
- Zwolak, M. and M. Di Ventra, Electronic Signature of DNA Nucleotides via Transverse Transport. Nano Lett., 2005. 5: p. 421-424.
- Smith, T., The hydrophilic nature of a clean gold surface. J. Colloid Interface Sci., 1980. 75: p. 51-53.
- Dunlap, D.D., et al., Masking generates contiguous segments of metal coated and bare DNA for STM imaging. Proc. Natl. Acad. Sci. (USA), 1993. 90: p. 7652-7655.
- Porath, D., et al., Direct measurement of electrical transport through DNA molecules. Nature, 2000. 403: p. 635-638.
- Fink, H.-W. and C. Schoenberger, Electrical conduction through DNA molecules. Nature, 1999. 398: p. 407-410.
- Kasumov, A.Y., et al., Proximity-Induced Superconductivity in DNA. Science, 2001. 291: p. 280-282.
- Lindsay, S.M. and M.A. Ratner, Molecular Transport Junctions: Clearing Mists. Advanced Materials, 2007. 19: p. 23-31.
- Cui, X.D., et al., Reproducible measurement of single-molecule conductivity. Science, 2001. 294: p. 571-574.
- Xu, B. and N.J. Tao, Measurement of Single-Molecule Resistance by Repeated Formation of Molecular Junctions. Science, 2003. 301: p. 1221-1223.
- Ohshiro, T. and Y. Umezawa, Complementary base-pair-facilitated electron tunneling for electrically pinpointing complementary nucleobases. Proc. Nat. Acad. Sci., 2006. 103: p. 10-14.
- He, J., et al., Identification of DNA base-pairing via tunnel-current decay. Nano Letters, 2007. 7: p. 3854-3858.
- He, J., et al., Hydrogen-bond electrical circuit for recognizing bases in single-stranded DNA. submitted., 2008.
Research in Chromatin and Biomolecular Structure
We would like to understand how genes work at a molecular level. Specifically, what is it about the binding of regulatory proteins (and other chemicals) that switches certain genes on and others off? It is here that the secret of development lies (i.e., what makes one cell a red-blood-cell and another a neuron?). and of the life-cycle of the cell.
The scanning probe microscopes, capable of imaging in fluid at very high resolution, offer the chance of imaging the components of a regulatory complex (DNA and protein) in natural conditions, and possibly even "in action" (that is, it might be possible to make movies of biological processes at the molecular level.
We have developed more sensitive instruments ("A magnetically driven oscillating probe microscope for operation in liquids" W. Han, S.M. Lindsay and T. Jing, Applied Physics Letters 69, 4111-4113 (1996)) capable of gentler imaging at higher resolution and we have used them to study structural transitions in DNA ("Kinked DNA" W. Han, S.M. Lindsay, M. Dlakic and R.E. Harrington, Nature 386, 563-564 (1997).)
We are currently working on two major projects using these instruments:
The first project involves examining kinking and bending in small DNA circles as they bind important proteins. The use of a circle simplifies interpretation of the bending, allowing us to identify "weak spots" along the gene ( see "Conformation and rigidity of DNA microcircles containing waf1 response elements for p53 regulation protein" H. Zhou, Y. Zhang, Z. Ou-Yang, S.M. Lindsay, X.Z. Feng, P. Balaguuurumoorthy and R.E. Harrington, J Mol Biol 306(2), 227-38 (2001)). This work is supported by the NIH and is part of a collaborative program with the Harrington Labs in the Department of Microbiology at Arizona State University.
The second project involves SPM methods to study chromatin remodeling. It is becoming increasingly clear that promoter chromatin structure and the remodeling of that structure in association with gene activation are crucial facets of eukaryotic transcriptional regulation. The recent development of an in vitro MMTV-LTR system (Hager G.L. "Understanding nuclear receptor function: from DNA to chromatin to the interphase nucleus." Prog Nucleic Acid Res Mol Biol 2000;66:279-305) that can reconstitute the correct promoter chromatin structure and the correct remodeling of that structure in vitro presents an unprecedented opportunity to study these important facets of transcriptional regulation. In particular, it will now be possible to study promoter chromatin with and without bound receptor and thus obtain information on this key first step of promoter recognition. We can then analyze the remodeled chromatin, to characterize the chromatin structure and transcription factor changes that have occurred as a result of remodeling.
Because of its scale of imaging, the atomic force microscope (AFM) is well suited for studying the various structural aspects of this process that we want to analyze: the linear organization (nucleosome locations), the higher-order structure (conformations of fully hydrated chromatin and transcription factors), and molecular recognition mapping (identifying specific molecules in the spreads based on antibody recognition). These approaches are a blend of established techniques and new AFM techniques that will be developed for this application but will undoubtedly prove useful for other biophysical and biological applications.
This project involves a collaboration with the labs of Dennis Lohr at A.S.U., the labs of Rodney Harrington at A.S.U., and the labs of Gordon Hager at the National Institute of Health. The collaboration also includes biophysicists Hansgeorg Schindler and Peter Hinterdorfer (University of Linz, Austria) pioneers in developing nm-scale molecular recognition techniques that use an antibody attached to an AFM probe.
- Specific Aims of this project include:
- Develop rapid and reliable antibody-based Molecular Recognition Mapping.
- Characterize and test Molecular Recognition Mapping on known and defined model chromatin.
- Carry out Linear Organization, Higher-Order Structures and Molecular Recognition Mapping Studies of LTR promoter region chromatin
- Study GR binding to this chromatin.
- Study chromatin remodeling in relation to Higher Order Structures and Molecular Recognition Mapping.
We are using both the STM and a novel STM/AFM combination instrument to probe electron transfer in single molecules. This is important for understanding processes such as photosynthesis and for building molecular electronic devices. It also touches on some interesting quantum mechanics!
To do this, we need to be able to make a variety of interesting molecules and tether them to a conducting substrate. We are working with Tom and Anna Moore and Devens Gust of the ASU Center for Early Events in Photosynthesis. We meet regularly to design new molecules, review our measurements and translate physics-speak into chemistry-speak and vice versa. Considerable effort is needed in pulling together the theoretical approaches of physicists and chemists, and one of collaborators (Otto Sankey) is a theoretical physicist working on this problem.
This work is funded by the Division of Biological Instrumentation of the NSF.
The tools behind the technology development are in the domain of engineering and chemistry: nanostructures that connect to molecules and instruments that manipulate and measure at the single-molecule level. The interactions and modeling come from physics: understanding the quantum phenomena that dominate interactions on this length scale and building simple models of the complex many-particle phenomena.
The systems come from biology: molecules involved in gene expression (signaling and the control of processes in living cells). Finally, the building of molecular model systems and the controlled assembly of nanostructures is dependent upon chemistry, biochemistry and materials chemistry).