The laboratory of Steven S. Smith, Ph.D. brings chemists and molecular biologists together for work on epigenetics as we help to build a science of bionanotechnology.
We approach epigenetics, like other problems in biology at the molecular level as a form of reverse engineering. Charles Darwin’s Evolution by Natural Selection, Adam Smith’s Unseen Hand, and Thomas Hobbes’ Leviathan provide the fundamental principle of biology: engineering by an unseen engineer.
In studies of epigenetics our efforts use reverse engineering to understand how patterns of methylation are set up on DNA and propagated in living things. In studies of bionanotechnology we study the science of supramolecular assembly in order to forward engineer molecular devices useful in biological research.
It is becoming increasingly clear that the new science of bionanotechnology will create a variety of devices that will not only improve existing approaches to biological research, but also augment existing medical diagnosis and treatment.
This new field uses information from molecular biology, chemistry and physics to link biological and non-biological molecules into complex bioassemblies not normally found in nature. In these applications, the goal of bionanotechnology is not only to produce the specificity and telemetry that a soft-landed radio beacon on the moon might exhibit, but also the capacity for detailed and serial analyses of the landing site that a soft-landed robot might exhibit.
No one expects that robotic control of a molecular device will be available anytime soon, but such a device might be preprogrammed to report several findings once its initial target was located. In developing nanoscale assemblies for these biological applications, we find that computer aided design is an essential step in the process. Devices now under development use the tools of computational chemistry (e.g. electronic structure calculation, homology modeling and molecular modeling) in addition to synthetic chemistry, and molecular biology.
The role of DNA methylation in biology is currently a topic of great interest in the research community. The study of cytosine methylation at CG sites in human DNA has received considerable attention. Those CG sites present in gene control regions have been especially well characterized. The study of CNG methylation in human DNA and the study of CG methylation outside gene control regions has received much less attention, even though these forms of methylation appear to make up the majority of methylation in human cells. In our lab we have been collaborating with scientists in Sweden and Moscow on what role, if any, that this sort of methylation may play in human cells.
To this end we are beginning to study the distribution of such sites in normal and cancer cells in order to better understand our findings with transgene-induced methylation at these sites human cells. It is a privilege to be able to work in such exciting fields and to be able to work with the group of talented M.D.s, postdoctoral fellows and Research Associates who have contributed to our work here at City of Hope. Our work is supported by the NCI postdoctoral fellowship program and their research granting program.
A major focus of our lab is the early detection of prostate cancer. Many recent studies show a change in the methylation status of specific genes in cancer cells. The level of methylation at CpG sites is measured by QPCR (see below). Using this method, detection of cancer has the possibility of being done faster and with less trauma to the patient. It also has the potential to be more accurate (more true positives and less false negatives) than with the PSA test, leading to more correct diagnosis and less unnecessary biopsies.
Our lab uses the Corbett Research Rotor-Geneä RG3000 to perform Quantitative PCR (QPCR). In our research with DNA methylation, we use Methylation Specific PCR (MSP) to determine the methylation status of CpG islands of genes thought to indicators of cancer. For this procedure, genomic DNA from a patient is treated with bisulfite to convert unmethylated cytosine residues into uracil. Methylated cytosine (5mCytosine) remains unreacted. Primers that distinguish between cytosine and uracil at CpG sites are used to amplify the target in separate reactions. Taqman probes specific to the bisulfite converted sequence between the primers allow for high specificity of detection by fluorescence.
The Agilent Bioanalyzer 2100 is a useful tool in the lab for quickly analyzing various DNA samples quantitatively. The DNA LabChipâ electrophoretically separates DNA based on size through microchannels in about 30 minutes with only 1ml of input. PCRs can be quickly checked for amplification of appropriately sized products or for the presence of primer-dimers. We have used the Bioanalyzer to develop an EMSA that can check for Protein-DNA complexes based on mobility shift.
A Waters HPLC System with a 996 Waters Photodiode Array Detector is used in the lab for oligomer (TAQMAN probes) purification. The purifications are performed on a PRP-1 column, using reversed-phase chromatography, in TeBAA (50 mM tetrabutylammonium acetate buffer, adjusted to pH 7.0 with acetic acid, in a gradient of acetonitrile), and TEAA buffer (50 mM triethylammonium acetate buffer, adjusted to pH 7.0 with acetic acid in a gradient of acetonitrile)
A Cyclone Plus DNA Synthesizer is used in the lab to synthesize oligos such as PCR primers, TAQMAN probes and substrates for enzyme kinetics experiments.
DNA and RNA quantification is done using a Nanodrop ND-1000 Spectrophotometer. “The NanoDrop® ND-1000 UV-Vis Spectrophotometer enables highly accurate analyses of extremely small samples with remarkable reproducibility. The patented sample retention system eliminates the need for cuvettes and capillaries which decreases the measurement cycle time. In addition, the high absorbance capability eliminates the need for most dilutions.” Quantifications are also performed using a Gilford Response spectrophotometer and a GeneQuant Pro RNA/DNA Calculator.
March 28, 2014
Information listed here is obtained from Pubmed, a public database; City of Hope is not responsible for its accuracy.