[Demelerlab] Proposed method -- next iteration

[Pahara, Justin (AAFC/AAC)]

Good Afternoon Borries,

Thank you for the feedback. I’ve included some comments/clarifications below:
I read through your document and am not at all convinced by your SELEX plan. The main problem is that you will not be able to "find" your antigen-bound aptamers of interest if you "train" them on a gemisch instead of a pure antigen. ssDNA will bind to anything, including RNA. In your step 1 experiment we have a whole bunch of labeled molecules (if the fluorophore actually survives the 95C denaturation cycle) which bind to anything in the lysate, potentially giving a very heterogeneous, non-specific mixture. Even if we have a fluorescent target to follow, we are clueless what all these different fluorescently labeled molecules are binding to. Potentially, each aptamer will bind to something else. How do you find the right one?

Agreed – the aptamer library which is comprised of a quadrillion possibilities will bind to many things, both naturally derived from E. coli, as well as *hopefully* to the engineered fluorescent protein. In the case of this mock experiment, the “right ones” would ultimately be enriched aptamers in a fluorescent protein fraction at the end of the SELEX rounds:


  1.  We do negative selection: Mix the aptamers with E. coli lysate to get rid of aptamers-E. coli derived interactions (the “heavy aptamer fraction”). You’re right, these will include aptamers binding to lots of E. coli stuff and so we want to get rid of those and keep the unbound fraction of “light aptamer fraction”. The assumption is that the “light fraction” of unbound aptamers do not interact well with E. coli stuff, however, some could interact with the fluorescent protein.
  2.  We enrich/amplify the pool of remaining unbound aptamers so we can do further selection rounds.

  1.  We then do positive selection: Mix the enriched aptamer library with engineered E. coli. The assumption is that the only biochemical difference from (1) is the presence of the engineered proteins. Therefore, we assume that any heavy bound aptamer fraction is interacting with the engineered proteins.
  2.  As you saw in the suggested protocol, we need to due several SELEX cycles of negative and positive selection and at the end, we hypothetically have an enriched pool of aptamers that specifically bind to the engineered proteins. We sequence these, order purified oligos, and test to confirm specific interactions.

For gathering the fractions, we don’t need to be precise. We simply need to have enough separation between bound/unbound fractions so we can aspirate, and then amplify with PCR (which is very sensitive and works in crude mixtures).


My proposal was different: pick a very specific and highly purified antigen target and perform your SELEX screen on that only. Once you got your DNA sequence narrowed down, fluorescently label the specific DNA molecule and mix it with the cell extract to see if it binds. Antigen is present = binding, antigen is not present = hopefully no binding. For that, the fluorescent approach will work fine, the nonspecific approach doesn't make sense to me. Maybe I am missing something?

I hope the above clarifies. We are hoping to achieve the same ending. The reason for suggesting that approach is that in the anaplasma infected blood samples there are few/no good targets for sensing it and we’ll need to find aptamers that bind to unknown anaplasma targets. The suggested experiment, is a first approximation of how we could do that in the anaplasma samples. If it doesn’t work in this mock experiment, then it is unlikely to work in our blood samples.

The experiment I propose is to first make a proof of concept: take a fluorescent protein (eGFP would be best) and see if we can train an aptamer sequence for eGFP using SELEX. Next, have one cell line that expresses eGFP, and another that doesn't, and then show that the DNA molecule actually binds to eGFP when it is endogenously expressed in a cell lysate.  If that works, we optimize the method to train an aptamer on your antigen of interest.

The limitation is that with anaplasma we ultimately do not have a good antigen of interest, so this proposed experiment has the potential to discover new ones whilst also identifying prospective aptamers.

Pls let me know what you think. I can drop the Uni to chat this or next week.

Best,
Justin.





On Wed, Jul 26, 2023 at 9:56 AM Pahara, Justin (AAFC/AAC) <justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>> wrote:
Good Morning Borries,

Just checking in to see if you’ve had a chance to check through the proposed SELEX, AUC method.

I hope your lab retreat was fun and energizing.

Best,
Justin.

From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Thursday, July 20, 2023 10:20 AM
To: Pahara, Justin (AAFC/AAC) <justin.pahara at AGR.GC.CA<mailto:justin.pahara at AGR.GC.CA>>
Subject: Re: Proposed method -- next iteration

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Hi Justin,
We are currently on our lab retreat in Montana and at a workshop, I'll get back to you next week in more detail.
Thanks for your patience, -Borries

On Thu, Jul 20, 2023, 08:32 Pahara, Justin (AAFC/AAC) <justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>> wrote:
Good Morning Borries, Amy,

Please see the attached document outlining the proposed experiment. Thank you for the back and forth discussion as it has helped us to better understand AUC, however, we still have much to learn 😊. Please let us know if there is anything that will not work.

I do have one remaining question, however, to see if we can do this entirely without a fluorophore:

If we are able to characterize the aptamers S value using absorbance UC, create an appropriate sucrose gradient, and then confirm the sedimentation rate of the unbound aptamers using absorbance UC again would we know with fairly high accuracy the position of the unbound aptamer fraction even in a whole cell lysate mixture? Therefore we would know the distance the fraction travels as a function of time and be able to crudely remove it? All without fluorescence?

@Borries, I know you’d prefer using a well defined and pure target, but I’m hoping we could run an experiment that reflects what we aim to do with the anaplasma in blood. If you feel the proposed method will not work, we are happy to pivot.


Thank you,
Justin.


Dr. Justin Pahara

Research Scientist and Project Lead

Nanotechnology (Biotic Stresses and Adaptation)
Agriculture and Agri-Food Canada / Government of Canada
justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>

Nanotechnologie (Adaptation et Contraintes Biotiques)
Agriculture et Agroalimentaire Canada / Gouvernement du Canada
justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>

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