The Konig vs Kaiser debate continues

And the Corruption Carries On.

For those following the DNA contamination story you are well aware of the Konig et al paper. This was followed by a paper from Kaiser (funded by DFG) claiming ‘nothing to see here’. Konig fired back with a list of flaws in the Kaiser paper, particularly pointing out the EtOH precipitations making their numbers meaningless.

Kaiser et al came back for more dirt knaps

Kaiser 2

They still failed to address the EtOH precipitation critique and the cost to prove that this is NOT an issue in their data is a few dollars. After being called out publicly about this fatal flaw in a paper Kaiser even cites, one can only assume they have performed the experiment and don’t wish to present it.

If you want a recap of this debate… This thread covers much of it.

But lets get into the latest Kaiser Paper that is wearing so thin on plausibility that its spawning memes over the authors incompetence (or complicity).

Their paper demonstrates in Figure A below that they are not using enough TritonX-100 or they need to use some heat (Speicher used heat and TritonX-100) to accelerate that reaction. They are on the front end of what could be an exponential curve and they refuse to address that.

Figure B is equally counter narrative. See how the measured DNA (Orange) barely moves as you spike double stranded Lambda DNA into the assay. That should tell you your DNA signals are being suppressed by the LNPs and TritonX and until you address that head on, no amount of RNaseA handwaving will address this. RNaseA is a good idea but those experiments in this context will all be confounded.

We have been using RNaseA and DNaseI since our first papers with qPCR and Qubits. They just are not doing a good job proving the TritonX (no heat) is opening the LNPs and enabling Qubit detection if their spike-in Lambda DNAs is suppressed on Qubit. This would a good time for them to consider Georgiou et al. who demonstrate that picogreen also under quantitates DNaseI treated DNA by 70% compared to the Lamda DNA used in this deception.

At least they admit to having a saturated system but lets carry on….

You have to ask yourself..

After Konig et al called Kaiser et al. out on their use of an EtOH prep, Kaiser is back with more data using the same flaw? This is because Kaiser knows if she performs a controlled EtOH precip with a 10bp ladder, she will not capture the small fragments and his numbers will begin to look like ours.

And No, we do not agree that DNA under 200bp is harmless once LNPs are involved and that DNA gets transfected. See Kwon et al. regarding the oncogenic nature of cytosolic DNA.

Particularly when the SV40 Enhancer is 72bp and is a known somatic hyper-mutability element (Senigl et al), binds to P53 (Drayman et al) and is a Nuclear Targeting sequence (Dean et al).

They didn’t just do this EtOH precip once. They did this twice! Before and after the enzyme treatments. They also did this in their last paper and when called out on it, continue to do it today.

They attempt to bring in LC-LC/MS to measure the DNA/RNA as an orthogonal check but perform an EtOH precip before this step as well (2 total). Instead of using DNaseI and RNaseA they cite a paper behind a paywall that suggests they are using Alkaline Phosphatase and Phosphodiesterase I (PDEI). PDEI won’t digest circular plasmids. These are likely a minority species in the sample if their linearization reaction went to completion. So this is likely a minor footnote in this discussion but not something we can ignore given the plasmids are replication competent with SV40 origins of replication.

They may also be inhibited by 1% TritonX-100 which is in the sample

Add in the Lipid Nanoparticles with 1% TritonX-100 and you have micelles that may recruit your co-factors for these enzymes. RNaseA doesn’t need a co-factor. DNaseI usually needs magnesium and some calcium.

So what we have is a study designed to sweep the results under the table by orders of magnitude. This alone could describe the difference between Konig and Kaiser. I personally think the number is between these two estimates based on our work and Kammermers work.

Being an inventor on some of the SPRI patents, I’m actually impressed that ChatGPT understands one of its key tricks. The magnetic beads are nucleating reagents and assist in precipitation of small molecules but you need to use divalent salts to get this right. NaCl will will fail to precip some small RNAs and DNAs. MgCl2 does a better job but you need 10-100mM amounts of it (not Molar like NaCl). This may not be very public information so I’m not surprised it didn’t know. If you push it, it corrects itself.

The Kaiser paper does a great job glossing over this overt deception by pointing to this very cool paper. Of course this paper doesn’t support the highlighted 100% recovery statement and never even measured it:)

It’s still a cool reference as it has great AFM of EtOH precips with various monovalent and divalent salts.

But Kaiser will never work with 100bp or 10bp ladders which are required for this application. The precipitation curves for smaller DNAs are very different and even ChatGPT knows this topic cold. If they can’t even keep up with ChatGPT, its not very impressive work. Its in fact deceitful.

The paper they cite claiming EtOH precips are 100% quantitative doesn’t look at DNA in this size size range. Smallest is from an 8Kb ladder which has no part number in the paper but usually kilobase ladders have 1kb,2kb,3kb,4kb,5kb,8kb, etc.. They usually don’t contain 10b-1,000 bp resolution.

Even with all this deception they still admit to being 1.8X over the limit, but they claim this is in range:) No discussion on how these LNPs, which make it so tricky to get nucleases involved and fowl up their measurements, are the same LNPs that make it difficult for your immune system to clear these nucleic acids. The 200bp limit was crafted under the assumption that the small DNAs will get digested once injected. True, if they are naked DNA but their paper very skillfully underscores how the LNPs need to be removed for these very clearance enzymes to work.

They have to invent new methods to detect and eliminate these nucleic acids once LNPs are involved. Same holds true for your immune system but they don’t want talk about that elephant in the room.

Interestingly enough the Journal Vaccine refused to send the Speicher paper out for review. They were happy to take the paper that was funded by the Vaccine developers at Deutsche Forschungsgemeinschaft. Even one with overt errors and shortcomings that have already been spoken about at length in other papers.

You read that correctly. Kaiser et al is funded by DFG who openly brags about developing this product. Is this made clear in the conflicts of interest statement on the paper? Clear as mud.

See how it makes this very bizarre statement 3 times like its conjuring up another meme lord?

“If there are other authors”?

Summary:

Its not hard to prove your EtOH precip is 100% quantitative for small fragments.

Just run your EtOH precip on a 10bp ladder and show us the yields before and after precip on Qubit or a gel. This is 60 minutes of work and there are reams of literature on DNA purification kits that make this fragment bias very clear. Given Kaiser et al reports to be on the Human RNAome Project Consortium, I find it really hard to believe they are this incompetent on the methodologies required to capture small RNAs.

There may be a funded reason Kaiser used an expensive LC-LC/MS as opposed this $10 control. Both their Qubit methods and their LC-LC/MS methods use an EtOH precipitation before them which is known to capture the large DNA/RNA and eliminate the small stuff. Puts the whole paper into the useless bucket.

Or useful for public deception.