Topo/DNA Complexes for Genomic Sequencing Studies
Topoisomerases are unique DNA binding proteins. They engage the helix in a series of transient breaking-resealing reactions to allow topological adjustments that arise from DNA templating. What is unusual is that these enzymes engage DNA in a short lived covalent mode. Specifically, the active site tyrosine for both type I and II enzymes, forms a phosphor-tyr link that is a covalent bond. The transient link evolved to ensure that these enzymes do not permanently damage the genome; thus, a tight coupling between the cleavage/relegation ensures that DNA discontinuities never accumulate. Interfacial Poisons (IFP) wreak havoc by stabilizing the discontinuity and trapping the Tyrosine-phosphor link in a covalent bond to the sugar phosphate back bone of DNA.
For detailed genomic studies, it would be useful to deep sequence all sites of action under the influence of a given IFP. We offer a procedure, the ‘TopoChIP’ to provide the end user with a known collection of topo/genomic DNA intermediates. These can be analyzed by modern illumina technology. Coupled with a clinical trial or for mechanistic consideration, this is a powerful approach.
Customers need only follow the lysing conditions, in their own lab (a single step), send us frozen lysates, and we will recover nearly pure topo/DNA complexes specific to a type I, IIα, IIβ event.
Please enquire about the details of this service. From this work flow:
- 1 Testing ´band depletions´ In this analysis we determine how well endogenous topo I or II is trapped onto genomic DNA. This is complementary to the In Vivo Link Kit. When cells are exposed to topo poisons, a fraction of the free enzyme becomes entrapped with high MW genomic DNA and cannot be extracted. This is reflected as a loss of band intensity of the nuclear extract, probed by Western blotting.
- 2 Genome wide association studies(GWAS) Determine(with high statistical significance and multiple reads) the target genes or intergenic regions.
- 4 Assess novel sites that are IFP specific There is ample evidence that some IFPs target different sequences in vitro; however, the physiological relevance of these findings in a cancer context is largely unknown.
- 5 Physiological background Tumor vs. normal cell contexts are likely to differ with regard to genomic sites; however this is not well understood.
- 6 Correlations between genomic action and tumor behavior What is the GWAS significance in benign vs. aggressive and normal cells? How is it different between drug congeners? This is a provocative idea that should be examined.
The Genomic studies are based on the In Vivo Link Kit (ICE Assays) This method can measure the endogenous action of topo in the tumor cell. The method is described (TG1021) and on a YouTube Video. The method is highly relevant clinically since it predicts the action of drug targeting in a cellular (chromatin) context. We direct the client on how to make the lysates and provide all required reagents. These extracts are then sent to us to prepare topo/DNA complexes for deep sequencing.
At TopoGEN, we developed, trademarked and patented many of the technologies associated with drug effects on DNA. We are leading experts at drug testing and assessment of drug action from a mechanistic standpoint. When you engage our services, we will describe and lay out a logical/rational approach that is rich in content and strongly mechanistic. We offer independent consultants who can validate our findings. Our services offer expediency and value as well. We can significantly accelerate the pace of your research, produce publication quality data and you will own the intellectual property that comes with our studies. We routinely enter into confidentiality agreements on projects, so your results are protected. Our in-house testing program is flexible and efficient. The following are important points associated with our services::
- 1 All positive and negative controls We demonstrate internally that all testing platforms are perfectly functional. As a result, there is no guessing. Your data will be clear and unambiguous. We stand behind our findings and certify the data.
- 2 All markers will be included To document and validate the results. Again, we certify tractable results that will be internally controlled.
- 3 Testing Flexibility We tailor each contract to offer maximum flexibility and coordinate with you over the course of the project to ensure intellectual flow of ideas and results. We will advise you on the best course of action with all hits. You can select the level of detail for the project that best suits your needs.
- 4 Over-run Flexibility In most if not all projects, there will be a need to re-examine, re-test or alter the course. How do we correct for work-load adjustments in the mid-stream? Our solution: we include on all projects, a small indirect “over-run fee“ that will allow us to perform additional key experiments without asking for additional funds. This is an advantage for clients and ensures that our high standards of data quality will be maintained. For example, if additional testing regimens are required, we carry out the service automatically. Importantly, there is no guessing on the budget and you will not pay additional fees.
- 5 Consulting We are a team of topo experts. All projects include email and direct SKYPE support over the course of the project. Our scientific staff will discuss and suggest future prospects or experiments.
- 6 Reporting For complete flexibility and to ensure cost-effective contracts, we offer two levels of reporting. At a basic level, included in all contracts, we provide well-documented data (powerpoint, PDF or password protected cloud account). We draw conclusions, presented as bullets, on each slide and make a recommendation for further development and maturation of the project. For clients who wish to have a & ´manuscript´ style report, we offer fully referenced publication style reports. These reports are professionally prepared and are suitable for presentation to regulatory agencies. These publication style reports can be reviewed independently by third party opinion leaders in the field and the reviews appended to the final report.
- 7 Hands-on Experience The company has been actively engaged in contract drug testing since the 1990s and we have the experience to get results that will enable ‘go/no go’ decision making on drug development.
- 8 Expedience In most cases, the project will be completed over a period of several business days. We will clearly stipulate how long the project will take and we deliver on time.
- 9 A Selection of Enzyme Targets We can test prokaryotic and eukaryotic systems with highly purified enzymes that are well-controlled reagents. We can provide additional documentation of purity and QC on all preparations. The following enzymes or tractable antibodies are currently in our inventory:
- Human DNA Topoisomerase I
- Human DNA Topoisomerase IIα
- Human DNA Topoisomerase IIβ (in vivo or ICE screens only, see next item below)
- 10 Advanced testing in living cells . TopoGEN is a pioneer in testing agents that operate in the context of the cell. For eukaryotic systems, we can perform rigorous testing in tissue culture cells. We offer a variety of cell lines suitable for topo I, IIα and IIβ testing. For example, we can test your drug (either as CIC or IFP) in virtually any cell line of interest.
- 11 The “Dual Gel Analytical System“ TopoGEN is unique in offering this service as part of our routine testing regimen. In all projects, we design our experiments to garner new information about your compound. For example, all gels are run in duplicate. One is a non-EB gel system, used to demonstrate unambiguous catalytic activity and the other gel is an EB containing gel to parse clearly the cleavage products. Since the reactions come from the same tube, we can be sure that the results are reproducible and accurate. Importantly, the non-EB gel system also reports whether the drug is a DNA intercalator, which may suggest genotoxicity in cells.