<div dir="ltr">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?<div><br></div><div>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.</div><div><br></div><div>Specifically:</div><div><div><ol start="1" type="1" style="margin-bottom:0in;margin-top:0in"><li class="m_3685040809722022969MsoListParagraph" style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif;color:rgb(68,114,196)">We do negative selection: Mix the aptamers with <i>E. coli</i> lysate to get rid of aptamers-<i>E. coli</i> derived interactions (the “heavy aptamer fraction”). You’re right, these will include aptamers binding to lots of <i>E. coli</i> 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 <i>E. coli</i> stuff, however, some could interact with the fluorescent protein.<u></u><u></u></li></ol><div><font color="#000000" face="arial, sans-serif">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?</font></div><div><font color="#000000" face="Calibri, sans-serif"><span style="font-size:14.6667px"><br></span></font></div><ol start="1" type="1" style="margin-bottom:0in;margin-top:0in"><li class="m_3685040809722022969MsoListParagraph" style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif;color:rgb(68,114,196)">We enrich/amplify the pool of remaining unbound aptamers so we can do further selection rounds</li></ol><div><span style="color:rgb(0,0,0);font-family:arial,sans-serif">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.</span><br></div></div><div><span style="color:rgb(0,0,0);font-family:Calibri,sans-serif;font-size:14.6667px"><br></span></div><div><ol start="3" type="1" style="margin-bottom:0in;margin-top:0in"><li class="m_3685040809722022969MsoListParagraph" style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif;color:rgb(68,114,196)">We then do positive selection: Mix the enriched aptamer library with engineered <i>E. coli.</i> 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.<u></u><u></u></li></ol><div><span style="color:rgb(0,0,0);font-family:arial,sans-serif">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?</span><br></div><div><font color="#4472c4" face="Calibri, sans-serif"><span style="font-size:14.6667px"><br></span></font></div><ol start="3" type="1" style="margin-bottom:0in;margin-top:0in"><li class="m_3685040809722022969MsoListParagraph" style="margin:0in;font-size:11pt;font-family:Calibri,sans-serif;color:rgb(68,114,196)">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.<u></u><u></u></li></ol><div><span style="color:rgb(0,0,0);font-family:arial,sans-serif">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.</span><span style="color:rgb(0,0,0);font-family:arial,sans-serif"> Can you explain that further?</span><br></div></div></div><div><br></div><div>-Borries</div></div><br><div class="gmail_quote"><div dir="ltr" class="gmail_attr">On Wed, Jul 26, 2023 at 1:48 PM Pahara, Justin (AAFC/AAC) <<a href="mailto:justin.pahara@agr.gc.ca">justin.pahara@agr.gc.ca</a>> wrote:<br></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div class="msg3685040809722022969">
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<p class="MsoNormal"><span style="color:rgb(68,114,196)">Good Afternoon Borries, <u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">Thank you for the feedback. I’ve included some comments/clarifications below:<u></u><u></u></span></p>
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<p class="MsoNormal">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?<u></u><u></u></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">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 *<b>hopefully</b>* 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:<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)"><u></u> <u></u></span></p>
<ol style="margin-top:0in" start="1" type="1">
<li class="m_3685040809722022969MsoListParagraph" style="color:rgb(68,114,196);margin-left:0in">
We do negative selection: Mix the aptamers with <i>E. coli</i> lysate to get rid of aptamers-<i>E. coli</i> derived interactions (the “heavy aptamer fraction”). You’re right, these will include aptamers binding to lots of
<i>E. coli</i> 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
<i>E. coli</i> stuff, however, some could interact with the fluorescent protein.<u></u><u></u></li><li class="m_3685040809722022969MsoListParagraph" style="color:rgb(68,114,196);margin-left:0in">
We enrich/amplify the pool of remaining unbound aptamers so we can do further selection rounds.
<u></u><u></u></li></ol>
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<li class="m_3685040809722022969MsoListParagraph" style="color:rgb(68,114,196);margin-left:0in">
We then do positive selection: Mix the enriched aptamer library with engineered <i>
E. coli.</i> 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.<u></u><u></u></li><li class="m_3685040809722022969MsoListParagraph" style="color:rgb(68,114,196);margin-left:0in">
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.<u></u><u></u></li></ol>
<p class="MsoNormal"><span style="color:rgb(68,114,196)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">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).<u></u><u></u></span></p>
<p class="MsoNormal"><u></u> <u></u></p>
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<p class="MsoNormal">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?<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">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.
<u></u><u></u></span></p>
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<p class="MsoNormal">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.<u></u><u></u></p>
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<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">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.<u></u><u></u></span></p>
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<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">Pls let me know what you think. I can drop the Uni to chat this or next week.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">Best,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:rgb(68,114,196)">Justin.<u></u><u></u></span></p>
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<p class="MsoNormal">On Wed, Jul 26, 2023 at 9:56 AM Pahara, Justin (AAFC/AAC) <<a href="mailto:justin.pahara@agr.gc.ca" target="_blank">justin.pahara@agr.gc.ca</a>> wrote:<u></u><u></u></p>
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<p class="MsoNormal">Good Morning Borries,
<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal">Just checking in to see if you’ve had a chance to check through the proposed SELEX, AUC method.<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal">I hope your lab retreat was fun and energizing.<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal">Best,<u></u><u></u></p>
<p class="MsoNormal">Justin.<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
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<p class="MsoNormal"><b>From:</b> Borries Demeler <<a href="mailto:demeler@gmail.com" target="_blank">demeler@gmail.com</a>>
<br>
<b>Sent:</b> Thursday, July 20, 2023 10:20 AM<br>
<b>To:</b> Pahara, Justin (AAFC/AAC) <<a href="mailto:justin.pahara@AGR.GC.CA" target="_blank">justin.pahara@AGR.GC.CA</a>><br>
<b>Subject:</b> Re: Proposed method -- next iteration<u></u><u></u></p>
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<span style="color:rgb(192,0,0)">CAUTION:</span><span style="color:black"> This email originated from outside of the organization. Do not click links or open attachments unless you recognize the sender and know the content is safe.<br>
</span><span style="color:rgb(192,0,0)">ATTENTION:</span><span style="color:black"> Ce courriel provient de l’extérieur de l’organisation. Ne cliquez pas sur les liens et n’ouvrez pas les pièces jointes à moins que vous ne reconnaissiez l’expéditeur et que vous
sachiez que le contenu est sûr.</span><u></u><u></u></p>
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<p class="MsoNormal">Hi Justin,
<u></u><u></u></p>
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<p class="MsoNormal">We are currently on our lab retreat in Montana and at a workshop, I'll get back to you next week in more detail.<u></u><u></u></p>
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<p class="MsoNormal">Thanks for your patience, -Borries <u></u><u></u></p>
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<p class="MsoNormal">On Thu, Jul 20, 2023, 08:32 Pahara, Justin (AAFC/AAC) <<a href="mailto:justin.pahara@agr.gc.ca" target="_blank">justin.pahara@agr.gc.ca</a>> wrote:<u></u><u></u></p>
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<p class="MsoNormal">Good Morning Borries, Amy,
<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal">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 <span style="font-family:"Segoe UI Emoji",sans-serif">😊</span>. Please let us know if there is anything that will not work.
<u></u><u></u></p>
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<p class="MsoNormal">I do have one remaining question, however, to see if we can do this entirely without a fluorophore:<u></u><u></u></p>
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<p class="MsoNormal">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?<u></u><u></u></p>
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<p class="MsoNormal">@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.<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
<p class="MsoNormal">Thank you,<u></u><u></u></p>
<p class="MsoNormal">Justin.<u></u><u></u></p>
<p class="MsoNormal"> <u></u><u></u></p>
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<p class="MsoNormal"><b><span lang="EN-CA" style="font-size:10pt">Dr. Justin Pahara</span></b><u></u><u></u></p>
<p class="MsoNormal"><b><span lang="EN-CA" style="font-size:10pt"> </span></b><u></u><u></u></p>
<p class="MsoNormal"><b><span lang="EN-CA" style="font-size:10pt">Research Scientist and Project Lead</span></b><u></u><u></u></p>
<p class="MsoNormal"><span lang="EN-CA" style="font-size:10pt"> </span><u></u><u></u></p>
<p class="MsoNormal"><span lang="EN-CA" style="font-size:10pt">Nanotechnology (Biotic Stresses and Adaptation)</span><u></u><u></u></p>
<p class="MsoNormal"><span lang="EN-CA" style="font-size:10pt">Agriculture and Agri-Food Canada / Government of Canada<br>
<a href="mailto:justin.pahara@agr.gc.ca" target="_blank"><span style="color:rgb(5,99,193)">justin.pahara@agr.gc.ca</span></a></span><u></u><u></u></p>
<p class="MsoNormal"><span lang="EN-CA" style="font-size:10pt"> </span><u></u><u></u></p>
<p class="MsoNormal"><span lang="FR-CA" style="font-size:10pt">Nanotechnologie (Adaptation et Contraintes Biotiques)</span><u></u><u></u></p>
<p class="MsoNormal"><span lang="FR-CA" style="font-size:10pt">Agriculture et Agroalimentaire Canada / Gouvernement du Canada</span><u></u><u></u></p>
<p class="MsoNormal" style="margin-bottom:12pt"><span lang="FR-CA" style="font-size:10pt"><a href="mailto:justin.pahara@agr.gc.ca" target="_blank"><span style="color:rgb(5,99,193)">justin.pahara@agr.gc.ca</span></a></span><u></u><u></u></p>
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