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


Brad Hall said...

Of course the idea is good. It is a sponsored projects. I want to address some wording and ideas though. Please address these within the text by either changing what you have written or providing more explanation within your abstract. Often this can be a few words or sentences. If I asked you to talk it through aloud, that would often be what you should write down.

1.) How does a protein "Follow the regulations of" anything? Reword to suggest more scientifically specific domains appear to function similarly to type III secretion pathways and provide a reference.

2. ) Nickel beads aren't a platform in themselves. Reword to suggest they are the preferred sieving process or something.

3.) What does the Sim reference detailing the genome have to do with your first paragraph? Explain your thought process or remove.

4.) I think paragraph two is a better intro paragraph. You may fit it in so the story goes from burk to disease to diagnostic protein target to aptamers.

5.) Paragraph 3 seems like fluff without much substance. More rhetoric than scientific writing.

6.) Reword the specific aim paragraph, second sentence starting with "It is thought..."

7.) Move and reword the final sentence of specific aim 2. What other conditions? Be "specific". This can change later.

Brad Hall said...

Oh, once you've corrected the paragraph, please let me know what changed in the comments so we can re-read...

Katherine Li said...

Ok, Done. But Brad, now my pictures have become black holes....

Gwen Stovall said...

You mention the protein has a His tag. The native protein does not contain a His tag. The recombinant protein purchased from the company contains a His tag ...

What does oligo dT offer you? How will this help your selection?

Also - are you concerned that you'll pull out ant-PVP aptamers? How did the Waterboer avoid this/

Katherine Li said...

This protein is synthesized by Kate Brown, so when we get it, it is functionalized with a 6X His Tag. The Oligo DT binds to everything so if the PVP works, it should show and difference between the + and the - selections, theoretically. We do - selections so I'm hoping to take out a lot of background using that. But also, after a few rounds, I will stop using the PVP and just do a few selections without it and then use a binding assay to determine how well PVP stops background binders. I hope to use the PVP as sort of a r0 pool starter that eliminates background to the tube and beads. This is just a theory I developed this summer.

Katherine Li said...

I have changed everything that you have suggested. Thanks for your input.

Brad Hall said...

Okay, for the pictures, just link to the picture online. I have corrected the text to remove the junk HTML formatting code. Please update the pictures or send the links to the ones you want via e-mail and I can post them for you.