We know exactly how and why the DNA is in the Moderna and Pfizer vials

And it shouldn't be, by the way -> cancer, cancer, bo-bancer

This past week has been epic for me. Not only did I have the extreme pleasure of volunteering alongside Kevin McKernan and Charles Rixey at Medicinal Genomics, but we have pretty much confirmed how the DNA is in the Moderna and Pfizer COVID shot vials.

Ages ago, when I was presenting the original findings that there was DNA in these vials, I was sleuthing how this happened by looking into the N1-methylpseudouridine modified RNA synthesis pathway as part of Process 2 manufacturing. Process 2 involved using a plasmid/E. coli system, don’t forget. And also don’t forget that this methodology was bait ‘n’ switched and was not safety tested.

N1-methylpseudouridine has a higher melting temperature than Uridine.¹ (Higher thermal energy or specific enzymatic activity is required to disrupt base pairing.) What this means in terms of it binding a cognate base is that it will require a very high temperature to rip them apart. Either that, or it will require a specific enzyme. Two examples of such specific enzymes are RNase-H (in us) and RNase-XT (on bench). It is well-known in nerdy science circles that DNase1 - the enzyme the COVID shot manufacturers used to chop up the DNA for endpoint synthesis cleaning - does not work on DNA:RNA hybrids.

DNA:RNA hybrids are something that occur in nature and in our cells, not just as part of some weird-ass modified mRNA in vitro transcription protocol to pump out arse-tons of material for LNP-wrapped experimental injectable products. We (our cells) have a specific enzyme called RNase-H to break them down because we sure don’t want accumulation of R-loops in our cells, because accumulation induces all sorts of disease states like cancer and amyotrophic lateral sclerosis type-4 (say that 5 times fast). By the way, R-loops are three-stranded structures: the DNA:RNA hybrid plus a single-strand of DNA displaced during hybrid formation.

By the way, since some of the DNA that we are detecting is SV40 promoter/enhancer, it is worth noting that it binds to p53.

Back to hybrids and how the DNA got in the vials in the first place. These hybrids formed as part of the production line and stayed in there because they weren’t cleaned out. This is more than a whoopsie, guys.

So what gives? How the hell did this happen? How is it possible that they a) didn’t care about finding excessive amounts of DNA in the vials,

and b) when they did, why the hell didn’t they make this connection to hybridization?

We did.

We also proved on the bench this week with minimal effort and cost that it is absolutely how the vials are all full of LNP-encapsulated DNA in addition to N1-modmRNA.

Our first qPCR run was almost too good to be true and like any good scientist, we doubled down (and back) and added additional steps/controls to our qubit assay to ensure that what we were seeing was real. What we were seeing in the qPCR results was a ton of SV40 in Pfizer vials LN2588, GK0936, and a ton of other DNA (ori and spike) in the Moderna vials O25G23A, AW4694B and AT0709B. The additional step was adding RNaseA (and DNase-XT), in addition to adding Triton X (to break up LNPs to release the DNA) and heating to 95 degrees (to ensure the RT enzymes were inactive in the case of qPCR quantitation) to ensure that RNA wasn’t masking the DNA signal as part of the qubit reads.

Kevin posted an excellent video of the qPCR results earlier today on X that you can watch by clicking on the photo below.

Please do watch it. It demonstrates that the spike DNA is protected from regular DNase I digestion (low Ct), while ori is not (high Ct). When we destroy the RNA:DNA hybrids (DNase-XT condition), the spike DNA now becomes sensitive and is also digested (Ct shifts right). This confirms that spike-sequence plasmid DNA fragments were present in RNA:DNA hybrids, presumably with the vaccine mRNA.

In terms of the video, the samples that contained RNA:DNA hybrids that were treated with DNase1 and DNase1-XT do reveal ori (plasmid backbone-associated) amplification curves successively farther to the right along the x-axis, meaning that there is less DNA (ct signal higher) early because in the case of ori, it is not protected by - or hybridized to - RNA.

We see more digestion occurring in the part of the plasmid that doesn’t have any RNA complement — i.e., non-spike regions. The spike region has RNA complement and the rest of the plasmid doesn’t, because of the design of the in vitro transcription workflow (spike sequence is transcribed into modified mRNA after linearization). So if you are the EMA and you target a qPCR assay against the KanR gene in the plasmid backbone, you’ll get a very high Ct or undetectable signal because that DNA was not hybridized to RNA and was therefore digested by the production DNase I. If, say, an independent lab targets the spike region, they will get a much lower Ct (higher residual DNA signal) because that portion was hybridized to mRNA, protected from the production DNase I, and therefore survives into the final product.

So Kevin took the injectable products across 5 lots (LN2588 (Pfizer), GK0936 (Pfizer), O25G23A (Moderna), AW4694B (Moderna), AT0709B (Moderna)) and put them in Triton X (to open up LNPs that “hide” the DNA) and then added DNase1 and DNase1-XT to the PCR preps. DNase1-XT ends the DNA:RNA hybrids.

So what would you expect? You would expect if the DNA:RNA hybrids were “protecting” the spike DNA from being digested by the enzymes, that the DNase1 and wouldn’t work on getting rid of those DNA:RNA hybrids, and that DNase1-XT would. This means that if spike DNA is protected or hybridized to RNA, we wouldn’t see spike move following the addition of DNase1, but we would see it move following the addition of DNase-XT. In the case of ori DNA, since it is not protected or hybridized to RNA, we would see it move (right along the x-axis) following the addition of DNase1, and even more so following the addition of DNase-XT because the enzymes can (progressively) effectively work on the ori DNA.

This is exactly what Kevin saw. The ori ct increased (farther right on x-axis indicating less DNA) respective to the enzyme (DNase1 or DNase-XT) in every sample because it got eaten by these enzymes. The spike ct didn’t change in the presence of DNase1 because (it’s not in the plasmid back bone and) it is protected by RNA as a hybrid. However, it did change (increased) in the presence of DNase1-XT. This confirms our hypothesis!

It means that the ori, as part of the plasmid back bone, ‘disappears’ as per our qPCR assay because it doesn’t have RNA protecting it. Spike on the other hand is resistant to DNase1 degradation so it sticks around. So the trick the regulators pulled is to only measure the stuff that is not hybridized and thus theoretically got chewed up by DNase1 as per their protocol during mRNA synthesis workflow. But this absolutely means that all those hybrids got left behind and they simply didn’t measure them, and we’ve now shown once again, that’s there’s a lot of DNA in these vials.

Do you see the trick? Just because you can’t see the cat doing yoga under the sheet doesn’t mean there’s not a cat doing yoga under the sheet!

This is more like detective work than lab work. It’s so very, very cool. Form a hypothesis, test it. Validate it. Solve problems.

I am waiting for Kevin’s write-up and will subsequently update this article as results come in.

Please read Sutton et al..

1. Parr CJC, et al. N1-Methylpseudouridine substitution enhances the performance of synthetic mRNA switches in cells. Nucleic Acids Res. 2020;48:e35. doi: 10.1093/nar/gkaa070