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Biosensors with Low Cross-reactivity for Waterborne Contaminants
Start Date 05/01/2007
End Date 04/30/2009
Primary Partner: Syracuse University
Primary Contact: Borer, Philip - Professor
Project Type: CARTI II
Technical Description:
This work will complete the 12-month project currently funded by CARTI, "A Direct Sequence Analysis (DSA) Method for Rapid Discovery of DNA Ligand Binding Sequences for Biosensor Technologies". As indicated in that proposal, this follow-up project will engineer and test AlloSwitch molecular switches specific for Cryptosporidium and for Giardia, and will generate low detection thresholds by linking multiple binding sequences. The currently funded project has succeeded in capturing DNA oligomers using the DSA method. DSA ends with the sequences being cloned into a plasmid. The new project will couple DSA to high throughput DNA sequencing to discover dozens of high affinity sequences from a starting pool of 107-1012 DNA or RNA oligomers. DSA will be a cheap and efficient method to capture high affinity sequences, tag their ends, and amplify for high throughput sequencing. The combined method will allow us to pick ligand binding sequences that are specific for Cryptosporidium or Giardia and that do not cross-react with other common components of public water supplies.
Expected Outcomes:
These dangerous organisms are resistant to chlorination so our sensors will provide a rapid and early warning of contamination that can cause debilitating illnesses that often kill infants, senior citizens, or immune-compromised individuals.
Accomplishments:
Aptamers are DNA/RNA molecules that have affinities for their targets similar to antibodies. Aptamers have shown great potential to replace antibodies in biosensors, point-of-care diagnostics, therapeutics, and all of the areas currently dominated by antibodies. Once aptamers have been identified, they can be produced for about 1% of the cost of antibodies. The commercial value of antibodies exceeds $40 billion per year. Sales of our proprietary aptamers may dominate antibodies within five years.
Commercial success has been limited by the cumbersome SELEX method for aptamer discovery. Only a few hundred aptamers have been discovered in the last 20 years compared to thousands of antibodies over the same period. We have proved that High Throughput Screening of Aptamers (HTSA TM) dramatically reduces the effort to discover new aptamers.
Our work continued to expand on HTSA discovery and validation. The techniques developed in the CARTI-I project and year one of CARTI-II laid the groundwork for what should become the primary method by which aptamers are discovered for thousands of biosensor targets: proteins, microorganisms, toxins, etc.
Work in the period from October 2008 through April 2009 included optimization of the HTSA method, its application to discovering aptamers specific for the surface proteins of cryptosporidium, and microarray methods for validation of protein-aptamer binding strength and specificity.
Benefits:
The use of biosensors (in this case Aptamers) for detecting water borne contaminants will provide a reliable method of maintaining high quality water at a fraction of the price of antibody technologies.
For more information:
http://www-hl.syr.edu/pressrelease/orthosystems.htm
http://cbbic.org/orthosystems.aspx
Publications:
A full PCT patent application was submitted Mar-2009: Borer, P.N. and McPike, M.P. (2009) Direct Selection of Structurally Defined Aptamers. International Application Number PCT/US09/037022 based upon some of the work conducted in both the CARTI-I and II grants.
A manuscript was submitted in Feb-2009: Kupakuwana, G.V., Chen L., Crill III, J. McPike, M.P. & Borer, P.N. (2009, submitted) Nature Methods: High throughput screening of aptamers. It has undergone several revisions, and discussions with the editor indicate that it will be accepted for publication in early 2010. The manuscript credits the CARTI grant.
Active grants that were stimulated by the CARTI grants include:
NIH R44 GM076811-03 to OrthoSystems, Inc.: “Microarray chips for rapid detection of high affinity nucleic acid sequences,” SMIR phase II $163k TC, 10/1/09 to 8/31/10. Mark McPike, PI.
NIH F31 GM086129-01 to Syracuse University,: “ Development of a Nucleic Acid Biosensor for Cryptosporidium parveum.” Ruth L. Kirschstein National Research Service Award predoctoral fellowship $161k TC, 8/15/08 to 8/15/12. Philip Borer, PI, Mento to Gillian Kupakuwana.
Pending grant applications stimulated by the CARTI grants include:
NIH R44 A1075739-02 to OrthoSystems, Inc,: “Simple DNA/RNA Probes for Protein Targets,” SBIR phase II $1,596k TC, 12/1/09 to 11/30/11. Philip Borer, PI. This grant received an outstanding priority scores and is likely to be funded.
NIH R43 XXyyyyyy to OrthoSystems, Inc,: “Aptamer Probes for Epigenetic Peptide Targets.” SMIR phase I $165k TC, 4/1/10 to 9/30/10, Mark McPike, PI.
Water contaminant analysis equipment
Image Credit: Orthosystems, Inc.; Community Based Business Incubator Center
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