Selection of RNA Aptamer against BPM-1027 for Diagnostic and Treatment Purposes of Burkholderia Pseudomallei

The full proposal is here.

Burkholderia Pseudomallei is a bacterium that is native to the Thailand area. There is not a great deal known about B. pseudomallei, but current research has shown that the incidents of infection are rising and resulting in more fatalities due to the slowness of diagnosis, exacerbated by the fact that meliodosis, caused by B. Pseudomallei, can mimic the symptoms of other illnesses such as tuberculosis (Falade, 2008)[1]. If left untreated, it would lead to septicemia in one or multiple organ systems (Hodgeson, 2009)[2]

BPS-1027 is a protein within B. pseudomallei that serves as an integral protein on the bacteria’s surface. Although the function of the protein is unknown, it has been discovered that it uses Type III secretion methods (Salamond, 1993)[3] to allow the proteins in the bacteria to be injected into the host cell via a needle-like structure, the distinctive characteristic of T3SS models. It is functionalized with a 6x Histidine tag, making Nickle-NTA beads the optimal filtering process for this selection (Ellington, 1990).

Specific Aim 1: Selection of RNA oligonucletides against BMP-1027 using Nickle-NTA beads.

BPS-1027 is a surface protein on B. pseudomallei that could theoretically be bound to by various RNA aptamers. Binding an aptamer to 1027 could cause a signal transduction that would allow for easy diagnosis and thus quick treatment of the patient. At the very least, the aptamer would serve as a better indictor of illness. The beads serve as the scaffolding for the protein and the RNA to interact and bind. A binding assay will be used to determine binding affinity. A doped pool will then be created to further test affinity.

Specific Aim 2: Usage of Polyvinyl Perolydine (PVP) to diminish background binding.

In selecting for an RNA aptamer, background binding often becomes an issue. Oligonucleotides often bind to the beads and even the side of the 1.7mL Eppendorf tubes. It is necessary to reduce the percentage of background binders in order to have a more pure pool of binders, specifically for the target, rather for the parameters surrounding the target. It has been shown that PVP, along with other solutions, can serve as a block against increased background binders (Waterboer, 2005)[4]. An 8% PVP solution will be utilized but concentrations that are at 10% and at 5% will also be tested. The PVP will be used in a selection series with Oligo-DT and the N34 Pool. Due to Oligo-DT’s high affinity to binding without specificity, the effectiveness of PVP on background binding can be more easily seen.

---

[1] Falade OO, Antonarakis ES, Kaul DR, Saint S, Murphy PA (2008). "Clinical Problem-Solving. Beware Of First Impressions". N Engl J Med 359 (6): 628–634.
[2] Hodgson K, Engler C, Govan B, et al. (2009). "A Comparison Of Routine Bench And Molecular Diagnostic Methods In The Identification Of Burkholderia Pseudomallei". J Clin Microbiol 47 (5): 1578–80.
[3] Salmond GP, Reeves PJ (1993). "Membrane traffic wardens and protein secretion in Gram-negative bacteria". Trends Biochem Sci 18 (1): 7–12.
[4] Waterboer T, Sehr P, Pawlita M (August 2005). “Suppression of non-specific binding in serological Luminex assays”. Journal of Immunological Methods 309 (1-2); 200-204