<div dir="ltr">Hi Justin,<div>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?</div><div><br></div><div>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?</div><div><br></div><div>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.</div><div><br></div><div>Amy, do you have a better idea?</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 9:56 AM Pahara, Justin (AAFC/AAC) <<a href="mailto:justin.pahara@agr.gc.ca" target="_blank">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>
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<p class="MsoNormal">Good Morning Borries, <u></u><u></u></p>
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<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>
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<p class="MsoNormal">I hope your lab retreat was fun and energizing.<u></u><u></u></p>
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<p class="MsoNormal">Best,<u></u><u></u></p>
<p class="MsoNormal">Justin.<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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<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,
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<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.
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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>
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<p class="MsoNormal">Thank you,<u></u><u></u></p>
<p class="MsoNormal">Justin.<u></u><u></u></p>
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<b><span lang="EN-CA" style="font-size:10pt">Dr. Justin Pahara</span></b><u></u><u></u></p>
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<span lang="EN-CA" style="font-size:10pt">Nanotechnology (Biotic Stresses and Adaptation)</span><u></u><u></u></p>
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<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>
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<span lang="FR-CA" style="font-size:10pt">Nanotechnologie (Adaptation et Contraintes Biotiques)</span><u></u><u></u></p>
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<span lang="FR-CA" style="font-size:10pt">Agriculture et Agroalimentaire Canada / Gouvernement du Canada</span><u></u><u></u></p>
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<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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