Nucleic Acid Aptamer Selection against Hcp-6 Surface Protein of Burkholderia pseudomallei for Diagnosis of Melioidosis

Nucleic Acid Aptamer Selection against Hcp-6 Surface Protein of Burkholderia pseudomallei for Diagnosis of Melioidosis
By Dara Goral

Abstract:

Burkholderia pseudomallei is a gram negative bacterium responsible for the disease, melioidosis, which has been the cause of many fatalities in Southeast Asia and Northern Australia[i]. Melioidosis is a septic disease that often causes abscesses in the spleen and liver[ii]. Because of the resilience of B. pseudomallei, many antibiotics have not proven effective in reducing infection and melioidosis is still associated with a high mortality rate of 40%. Most fatalities occur within the first 48 hours of infection and it is difficult to diagnose this disease in that short amount of time[iii].

                  An approach is introduced that seeks to bind short RNA oligonucleotides to the Hcp-6 surface protein of Burkholderia pseudomallei. This surface protein is an excellent marker because it is a major virulence determinant for the melioidosis. In the suggested approach, the use of RNA ligands, called aptamers, prove to be a promising method for binding of Hcp-6 because of successful aptamer selections against many other proteins with various functions. Aptamers are powerful tools in diagnostics and drug delivery due to their high specificity and affinity for protein targets. In finding an aptamer with a high affinity for Hcp-6, it could be a crucial step in the development of a method for diagnosing melioidosis.

Specific Aim 1: Perform SELEX (Systematic Evolution of Ligands by Exponential Enrichment) in order to select an aptamer to bind to the Hcp-6 surface protein of Burkholderia pseudomallei.

A highly specialized aptamer may be developed that binds with great affinity for this surface protein allowing for easy identification of this bacteria in any sample.

Specific Aim 2: Attach a fluorescent tag to the RNA aptamer and experiment use of detection.

The attachment of a fluorescent tag would allow for easy detection of Burkholderia pseudomallei in a given sample. This could expedite the process of diagnosing melioidosis.

Figure 1: The RNA aptamer will bind to the Hcp-6 surface protein of the bacterium. After this specific aim 1 is achieved, a fluorescent tag will be attached to the aptamer for detection.

Previous Research: Two rounds of selection have already been completed against this target.

Purchasing Information: This target has already been obtained from the Katy Brown Lab.

Target Proposal
Progress Report 1
Progress Report 2
Final Manuscript

References:

[i] Brett, P. J., D. DeShazer, and D. E. Woods. (1998) "Burkholderia Thailandensis Sp. Nov., a Burkholderia Pseudomallei-like Species." International Journal Of Systematic And Evolutionary Microbiology 48.1: 317-20.

[ii] Currie, Bart J., Dale A. Fisher, Nicholas M. Anstey, and Susan P. Jacups. (2000) "Melioidosis: Acute and Chronic Disease, Relapse and Re-activation." Transactions of the Royal Society of Tropical Medicine and Hygiene 94.3: 301-04.

[iii] Rajchanuvong, A., W. Chaowagul, Y. Suputtamongkol, M.d. Smith, D.a.b. Dance, and N.j. White. (1995) "A Prospective Comparison of Co-amoxiclav and the Combination of Chloramphenicol, Doxycycline, and Co-trimoxazole for the Oral Maintenance Treatment of Melioidosis." Transactions of the Royal Society of Tropical Medicine and Hygiene 89.5: 546-49.