[Demelerlab] Proposed method -- next iteration

[Borries Demeler]

Since aptamers will bind to many different molecules, this will include
small positively charged molecules as well as many bigger things. We will
get a broad spectrum of different molar masses for the complex, some
complexes that are almost the same size as the free aptamers, ranging to
others that are much bigger. How do you know which size is the right one?
If you separate based on free and some complexed MW that is easy to
separate, how do you know that you don't completely miss the relevant
complex because it is smaller than what you can separate from free aptamer?

It sounds like you expect to be able to tell the difference by doing your
screening somehow by comparing lysates/serum from infected vs. negative
control samples. This part is what I can't follow right now. How are you
telling which aptamer is bound to the "right" antigen? Sorry, but if I
cannot understand the downstream approach, I cannot design the right design
for this experiment. I need to gain a better understanding first.

Specifically:

   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.

So you hypothesize that some aptamers will bind only to Ecoli and therefore
you can remove them from the pool by telling the fractions that sediment
faster than unbound aptamer, correct? Once you amplify the remaining pool,
you will not introduce further variability? If you did, you would be back
to square one. If you don't, how do you have enough variability left?


   1. We enrich/amplify the pool of remaining unbound aptamers so we can do
   further selection rounds

Whatever is left, may just bind weakly to your target, and you are back to
square one, because it may bind just as weakly to some other proteins.


   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.

So your hope is that whatever aptamer is left from your negative screen
contains the one aptamer that actually can bind to your target, and
strongly?


   1. 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.

So how do you plan to get around the heterogeneity that starts with very
small molecules binding to the DNA aptamers that sediment at almost the
same rate as unbound aptamers? What about the question about heating a
fluorescently labeled DNA molecule to 95 degree and hoping the fluorescence
survives, has that been tested? So I guess your plan is for us to first
measure unbound aptamer in a sucrose gradient to find out how fast it is
sedimenting so you can separate unbound aptamer from everything else by
running the same gradient, correct? I just don't understand how you could
come up with specificity if you don't even know what you plan to bind with
your aptamer by this approach. I don't understand the basis by which you
are separating the "correct" complex from all the others that will form if
you don't even know what the correct complex looks like. Can you explain
that further?

-Borries

On Wed, Jul 26, 2023 at 1:48 PM Pahara, Justin (AAFC/AAC) <
justin.pahara at agr.gc.ca> wrote:

> 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> 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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