[Demelerlab] Proposed method -- next iteration

[Borries Demeler]

Hi Justin,
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?

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?

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.

Amy, do you have a better idea?

-Borries

On Wed, Jul 26, 2023 at 9:56 AM Pahara, Justin (AAFC/AAC) <
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>
> *Sent:* Thursday, July 20, 2023 10:20 AM
> *To:* Pahara, Justin (AAFC/AAC) <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> 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
>
>
>
> Nanotechnologie (Adaptation et Contraintes Biotiques)
>
> Agriculture et Agroalimentaire Canada / Gouvernement du Canada
>
> justin.pahara at agr.gc.ca
>
>
>
>
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