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IVT and mRNA Purification
Trial Yeast mRNA LNPs
</p>
<pid="Yeast-datetime">
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09/07/24 - 30/08/24
26/06/24 - 16/09/24
</p>
<ulid="Yeast-participants">
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<li>Adrian</li>
<li>Henriete</li>
<li>Kate</li>
<li>Lea</li>
<li>Thijmen</li>
<li>Maks</li>
</ul>
<divid="Yeast-journal">
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<tableclass="borderless">
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<tr><tdclass="title-border">
<h2>RAD112: IVT of the GFP and Factor VIII RNA</h2>
<p>
<b>Background:</b> The
"<ahref="https://static.igem.wiki/teams/5342/documents/protocols/in-vitro-synthesis-of-rna-using-t7-rna-polymerase.pdf"target="_blank">In vitro synthesis of RNA using T7 RNA polymerase</a>”
protocol was used to translate mRNA from the cpFVIII DNA samples received in the experiment RAD110, as well as the GFP DNA received in the experiment RAD109.
<b>Background:</b>To understand how the formulation of the LNPs go, an experiment without using LNPs at first must be conducted.
LNP preparation followed by the “<ahref="https://static.igem.wiki/teams/5342/documents/protocols/lnp-procedure.pdf"target="_blank">LNP procedure</a>”.
Ionizable lipids were formulated in RAD201.
Cholesterol, PBS buffer, and compounds to make citrate buffer 10mM pH4 are present.
The compounds DOPC & DOPE - 50 ul of 50 mg/ml; DSPE PEG - 2000 - 50 ul of 50 mg/ml have been received from the Zainab Javed.
mRNA should be stable in citrate at 4 pH [1]. At high pH, mRNA is not comfortable.
Yeast mRNA used: torula yeast RNA.
</p>
<p>
<b>Methods:</b>
</p>
<p>Ethanol injection - provides low mRNA encapsulation efficiency, but is cheap and fast. [2]</p>
<p>DLS - size and Z-potential</p>
<p><b>Calculations:</b></p>
<p>
1 attempt:
250 uL liposomes, 10 ul DOPE, 1 - 2 ul charged lipids, 1uL chol - 7 day
(half of these for LNPs)
</p>
</td></tr>
<tr><tdclass="journal-td">
<p>9th of July 2024</p>
<p>26th of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Lea</li>
<li>Adrian</li>
<li>Kate</li>
</ul>
<h4>Experimental:</h4>
<ulclass="emptylist">
<li>The samples DSPE PEG - 2000, DOPC, & DOPE were dissolved in chloroform, which we have evaporated.</li>
<li>In around 30 minutes, DOPC evaporated under N2.</li>
<li>These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry.</li>
</ul>
</td></tr>
<tr><tdclass="journal-td">
<p>27th of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
<li>Kate</li>
</ul>
<p>The next day they were resuspended in 50 ul of ethanol, concentration - 50 mg/ml for DOPC and DOPE, and 10 mg/ml of PEG.</p>
</td></tr>
<tr><tdclass="journal-td">
<p>28th of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
<li>Kate</li>
</ul>
<ulclass="emptylist">
<li>The samples were nitrogen blown for 15 seconds to remove oxygen traces.</li>
<li>Then the samples were sonicated for 5 minutes.</li>
<li>Citrate 10mM was prepared by the procedure listed in the “<ahref="https://static.igem.wiki/teams/5342/documents/protocols/lnp-procedure.pdf"target="_blank">LNP procedure.</a>"</li>
</ul>
</td></tr>
<tr><tdclass="journal-td">
<p>29th of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
</ul>
<p>I weighed 1.5 mg and added it to 70 ml of 10 mM, 4 pH citrate solution.</p>
<li>In around 30 minutes, DOPC evaporated under N2.</li>
<li>These samples were vacuumed in a desiccator chamber for 15 minutes, and then left overnight to dry.</li>
</ul>
</td></tr>
<tr><tdclass="journal-td">
<p>30th of August 2024</p>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
</ul>
<h4>Experimental:</h4>
<ulclass="emptylist">
<li>I added the LNP sample to fill the whole volume of the membrane chamber, and let it centrifuge for 15 minutes, 5000 g, RT. (I through out salts)</li>
<li>I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts)</li>
<li>Again, I added 10xPBS in the space which got freed up by the removed salts, and then let it sit in the centrifuge for 30 minutes, 5000 g, RT. (I collected salts)</li>
<li>I did not add PBS and just let the left sample run in the centrifuge for 30 minutes, 5000 g, RT. (No salts were present.)</li>
<li>LNPs1 were collected.</li>
<li>I analyzed the samples with a DLS machine</li>
</ul>
</td></tr>
<tr><tdclass="journal-td">
<p>3rd of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
<li>Maks</li>
</ul>
<ulclass="emptylist">
<li>Solution of purified LNPs1 turned out purple today.</li>
<li>In two 1.5 ml membranes, in each, around 1 ml of sample was inserted, and then filled up with a 10x PBS buffer.</li>
<li>Then, the samples were weighed, and inserted into a centrifuge for an hour at 5000 g, RT.</li>
<li>Retained mixture was dissolved up to 1500 ul & the elution was also collected.</li>
</ul>
</td></tr>
<tr><tdclass="journal-td">
<p>5th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
</ul>
<ulclass="emptylist">
<li>Solution of unpurified LNP (2x1.5ml) pipetted into membrane opening of 2 centrifugal ultrafiltration tubes. </li>
<li>Tubes are filled with a 10x PBS buffer until the volume reaches the level of the bead of the tube.</li>
<li>Tubes weighed to be within 0.6g of each other and centrifuged for 1 hr at RT at maximum speed (3750 RPM).</li>
<li>LNPs2 were collected.</li>
<li>On the next day, solution LNPs2 also turned purple.</li>
<p>Figure 4. DLS. Z-potential measurement of LNPs1 in the PEG2000 material mode; Dispersant PBS</p>
<p>
The results above show that our LNP size is around the desirable 100 nm. However, it is a bit more, it is assumed that it is due to the ethanol injection methods uncertainty, so this could be improved with microfluidic mixing.
</p>
<p>
The Z-potential showed weird results. This is assumed to be related to the high acidity of the sample due to its improper purification - always add the sample only to the highest mark in the membrane chamber, and the rest is buffer. This is probably the reason why some ions and salts did not go away.
</p>
<p>
Solution of purified LNPs1 turned out purple after several days. This is assumed due to a highly acidic environment, which also proves the above point.The LNPs were not sonicated, and this is why they showed bigger size. The purification method was changed, so it is strange that the LNPs still showed high pH. It potentially might be due to not sufficient dialysis steps (only one hour).
</p>
<p><b>Conclusion:</b> LNPs have turned their color to purple, which suggests that either some reaction with LNPs occurred during their storage, or some contamination happened. LNPs were stored in RT, now they will be stored at 4C. LNPs were taken up by syringe during the dialysis analysis. Now, an aliquot will be taken for the analysis, since there is a suspicion that metal from the syringe might have colored the sample.</p>
<p>Always add the sample only to the highest mark in the membrane chamber, and the rest is a buffer. Also, samples should be purified for more than 1 hour. (Potentially, the PBS buffer should be not 10x, but 1x). The Z-potential was most probably affected by purification.</p>
<p>Finally, sonication should always be performed before the DLS measurement. </p>
<p><b>Advice:</b> always sonicate LNPs before analysis/usage, since they agglomerate</p>
<p><b>References:</b></p>
<ol>
<li>
a mastermix for 10 reactions of 20uL was prepared following the protocol for IVT. → ! Guanosine-5’-monophosphate was not used in the reaction mix, because it prohibits capping.
mastermix amounts:
<ul>
<li>Tris (1M): 8uL</li>
<li>MgCl2 (1M): 5uL</li>
<li>DTT(0.1M): 10uL</li>
<li>spermidine (25mM): 8uL</li>
<li>rNTPs(20mM): 40uL</li>
<li>MilliQ: 129uL</li>
<li>total: 200uL</li>
</ul>
</li>
<li>prepare 10 epps, with each 20uL mastermix</li>
<li>1uL dsDNA of the five different DNA samples (4 FVIII DNA, 1 GFP DNA) was added to epps (for FVIII linearised plasmid, 75 ng of DNA is needed for 20uL reaction, for GFP it is 26ng. The concentration of the FVIII DNA was close enough that 1uL could be taken from the tube. The GFP DNA was diluted 1:1 to a concentration of 30ng/uL and 1uL was used from that )</li>
<li>For every different DNA sample, one epp got 2uL T7 polymerase and the other 4uL. → 2uL is the amount recommended by the protocol, but because of the length of the FVIII gene, we wanted to see if a larger amount of T7 would be beneficial.</li>
<li>The reaction was incubated at 37 C for 3 hours.</li>
<li>5uL of all samples was mixed with 2uL loading dye and run on an agarose gel (110V 40 min)</li>
<li>Hamatani, Kiyohiro & Eguchi, Hidetaka & Takahashi, Keiko & Koyama, Kazuaki & Mukai, Mayumi & Ito, Reiko & Taga, Masataka & Yasui, Wataru & Nakachi, Kei. (2006). Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. 54. 773-80. 10.1369/jhc.5A6859.2006. </li>
<li>Jung, Han Na, et al. “Lipid Nanoparticles for Delivery of RNA Therapeutics: Current Status and the Role of in Vivo Imaging.” Theranostics, vol. 12, no. 17, 2022, pp. 7509–7531, https://doi.org/10.7150/thno.77259.</li>
<li>“Cell Press: STAR Protocols.” Cell.com, 2024, star-protocols.cell.com/protocols/3495#step-by-step-method-details.</li>
<p>Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD203: Formulation of LNPs with Torula Yeast mRNA inside, second attempt</h2>
<p>
It is weird that 1, 2 and 4 with 2uL T7 did not show a lot of transcription while 3 seems to have a lot, because they should practically be the same. Overall, the reaction seems to work better with 4uL T7.
The FVIII RNA was expected to be longer than 7000 instead of 1200. This could be the result of RNA degradation or self splicing, but it could also be because of the properties of the gel and because RNA is not linear.
In addition, the GFP RNA did not work out that well in both cases. it could have something to do with the template. maybe the calculations on the amount of template needed was incorrect.
<b>Background:</b> The LNPs from the RAD202 experiment have turned out the needed size, but have changed color, and showed weird Z-potential.
The goal of RAD203 is to figure out why this might be happening, as well as trying to introduce a new analysis method - encapsulation analysis using fluorescence spectroscopy of LNPs. The protocol:
<li>Ethanol injection - provides low mRNA encapsulation efficiency, but is cheap and fast. [2]</li>
<li>Encapsulation efficiency - The amount of encapsulated mRNA was calculated by subtracting the amount of free mRNA from the total mRNA. Encapsulation efficiency was determined by dividing the amount of encapsulated mRNA by the amount of total mRNA. [3]</li>
<li>DLS - size and Z-potential</li>
</ul>
<p><b>Safety: SYBR® Gold - Mutagenic; dissolved in buffer - keep in fridge, -4C (months); regular - in freezer, -20C.</b></p>
</td></tr>
<tr><tdclass="journal-td">
<p>3rd of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Maks</li>
</ul>
<h4>Experimental:</h4>
<p>
→ I (Lea) recalculated the amount of template needed and I did receive a different outcome: 45 ng/20uL. This is a 15ng difference from what was used in this experiment.
Compounds were mixed together in the same way as RAD202.
NOTE: Yeast mRNA in citrate solution was left out of the fridge overnight.
</p>
<p><b>Conclusion:</b> Experiment needs to be repeated with proposed changes:</p>
</td></tr>
<tr><tdclass="journal-td">
<p>9th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>different version of T7 poly.</li>
<li>Add RNAse inhibitor.</li>
<li>Reduce temperature to prevent autocatalytic breakdown.</li>
<p>Figure1. NOTE: Upon storage at -4C, and not interacting with DLS metallic syringe, the LNPs have not turned purplish! </p>
<p>Encapsulation efficiency analysis</p>
<p>Add 0.24 ul of LNPs (stock - 147.5 g/ml) to 10 ml of solvent (PBS 1x) -> 0.0036 g/ml LNPs</p>
<p>Yeast mRNA (was stored at -20C) was warmed to liquid.</p>
<p>The following delusion scheme was prepared:
6 samples dissolve each 2-fold:</p>
<tableclass="journal-table">
<tr>
<td></td>
<td>Dilusions: mg/ml</td>
</tr>
<tr>
<td>1</td>
<td>0.01455476731</td>
</tr>
<tr>
<td>2</td>
<td>0.007277383655</td>
</tr>
<tr>
<td>3</td>
<td>0.003638691828</td>
</tr>
<tr>
<td>4</td>
<td>0.001819345914</td>
</tr>
<tr>
<td>5</td>
<td>0.0009096729569</td>
</tr>
<tr>
<td>6</td>
<td>0.0004548364784</td>
</tr>
</table>
</td></tr>
<tr><tdclass="journal-td">
<p>11th of July 2024</p>
<h4>Participants:</h4>
<ul>
<li>Kate</li>
</ul>
<p>DLS analysis</p>
<ulclass="emptylist">
<li>The LNPs were taken out of the freezer, and then transferred to another vial with glass pipette. NOTE: LNPs were not purple after withdrawal from the fridge.</li>
<li>LNPs were NOT sonicated before the measurement.</li>
<li>LNPs were then sonicated, and measured twice in size, and only one Z-potential measurement (B, C measurements).</li>
<li>LNPs have turned purple after a while after the first measurement</li>
<p>Figure 0. Purple color change observed after DLS. Reason Unknown. Potentially - metal on the needle/ processes that happen within a DLS machine.</p>
<p>Another aliquot of LNPs was taken, sonicated and analyzed on DLS (D).</p>
<p>Figure 3. DLS after sonication. Size measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS; DLS after sonication (top 3). Z-potential measurement of LNPs2 in the PEG2000 material mode; Dispersant PBS (middle one); DLS after sonication. Size measurement of LNPs2 in the Liposomes material mode; Dispersant PBS (bottom 3)</p>
<p>After DSL LNPs turn purple for unknown reasons. In the 1st measurement RAD203 I did sonicate LNPs (helps agglomerate them), so I measured them again after sonication, and received too big of sizes of LNPs which proved that something changes them after DLS and purple color is the consequence.</p>
<p>The LNPs in the first DLS measurement were not sonicated, which gave results with a bigger size. They were then sonicated after the measurement, and taken for a second DLS measurement.</p>
<p>After the first DLS measurement, the LNPs have turned purplish, and gave weird results in the second DLS measurement.</p>
<p>Encapsulation efficiency</p>
<p>Analysis was not successful, potentially due to the procedure performed in a rush. The background noise value was for some reason showing values bigger than the measured values, which of course can not be the case.</p>
<p><b>Conclusion:</b> The LNPs hich are measured for the second time after DLS show weird change to purple color, and potentially form conglomerates, since the size values of LNPs in the second DLS increased significantly. </p>
<p>Z potential still behaves weirdly. It is assumed that the following behavior might be due to the low pH in the LNP solution which makes ionizable lipids gain positive charge, so in the next experiment, 1xPBS with higher pH (~7) will be used to check if Z will change.
Encapsulation efficiency analysis will be performed more carefully in the following experiments, more time will be provided for the procedure to prevent potential mistakes.
</p>
</td></tr>
<tr><tdclass="title-border">
<h2>RAD204: Formulation of LNPs with Yeast mRNA inside, second attempt</h2>
<p>
<b>Background:</b> The purplish color behavior was related to the DLS - procedure. However, the Z-potential from the experiment RAD 202-203 can still not be changed. This is why, in this experiment PBS1x will be used instead of PBS10x to see if this affects the Z-potential.
</p>
<p>Yeast mRNA used: torula yeast RNA.</p>
</td></tr>
<tr><tdclass="journal-td">
<p>13th of September 2024</p>
<h4>Participants:</h4>
<ul>
<li>Adrian</li>
<li>Kate</li>
</ul>
<h4>Experimental:</h4>
<ulclass="emptylist">
<li>The LNP beaker was cleaned with ethanol & then acetone.</li>
<li>
Lipids were sonicated for 5 min at RT, and formulated in LNPs with following proportion:
<table>
<tr>
<td></td>
<td>Added ul</td>
<td>Theory ul</td>
</tr>
<tr>
<td>ionizable lipids</td>
<td>3.88</td>
<td>3.881271282</td>
</tr>
<tr>
<td>DOPE</td>
<td>2.325</td>
<td>2.325207452</td>
</tr>
<tr>
<td>cholesterol</td>
<td>5.24</td>
<td>5.236352597</td>
</tr>
<tr>
<td>PEG-lipid</td>
<td>3.259</td>
<td>3.258548536</td>
</tr>
<tr>
<td>Ethanol</td>
<td>985</td>
<td>985.2986201</td>
</tr>
</table>
</li>
<li>Dialysis tubes were cleaned after experiment RAD203, LNP, cleaning of dialysis tubes.</li>
<li>I added the mixture of lipids 1 ml to 3 ml mRNA in a citrate solution with a pasteur pipette dropwise.</li>
<li>Lipids were mixed together for 10 (+-2) min.</li>
<li>I have dialysed the LNPs at 3270 g for 1 h in PBS1x, then resuspended them in PBS1x, and set them for dialysis for another hour.</li>
<li>The LNPs were suspended up to 1.5 ml PBS. Total volume - 3 ml. </li>
<p>Figure 1. Agarose gel electrophoresis of IVT samples. To prevent waste, we use the same agarose gel for multiple experiments. The crossed out lanes are from a previous experiment.</p>
<p>Figure 2. DLS Z-potential measurement of LNPs204 in the Liposome material mode; Dispersant PBS</p>
<p>In this experiment, LNPs were sonicated before the measurement, so the size of theirs is as expected - around 100 nm [1].
However, the Z-potential was still showing weird numbers - high positive which means that particles are very dispersed in the mixture, but for some reason positively charged.
It is assumed that these particles are behaving this way because the LNP composition is different from [1]. PEG-2000 is used, and no mannose PEG is present. It is assumed that the mannose must provide LNPs with some sort of shielding effect, negating their positive charge.</p>
<p><b>Conclusion:</b> The Z potential seems to be high due to high dispersion of the LNP particles, and high which is a difference from the reference [1] possibly due to different LNP composition.
This means that PBSx1 or PBSx10 do not affect the Z, but the properties of particles.
