RNase H and DNA:RNA hybrids

The R-Loop hypothesis

One of the reasons we believe DNaseI is failing to fully digest the DNA in the modRNA vaccines is because the IVT reaction that makes modRNA from dsDNA templates (plasmids) creates molecules that are DNA:RNA hybrids.

This has DNA on one strand and RNA on the other. DNaseI doesnt recognize these molecules and when you have a vast excess of RNA that is complementary to a DNA template the DNA will base pair with the RNA instead of its complement DNA strand which is in much lower quantity than the RNA.

We covered this in a former substack but until now it was all hypothetical.

One of the work horses of genomics is an enzyme known as RNaseH which stands for RNase Hybrids. This is an enzyme commonly used to make cDNA libraries as it will erase the RNA once cDNA is synthesized from an RT polymerase.

Since we frequently make RNA-Seq libraries for sequencing Hop Latent viroid infected cannabis we happened to have some of this enzyme in the freezer and I simply applied it to vaccine mRNA while exploring its impact on purely circular HpLVd RNA (Hop Latent Viroid).

In this experiment we are using a ssRNA specific dye known as AccuBlue from Biotium. This is a similar dye to RiboGreen from Invitrogen. We can see 108-120 ng/ul of RNA in the vaccines before a 10 minute treatment with 2ul this enzyme. Once treated the RNA signal drops ~50%.

When this enyzme is used on pure circular RNA (HLVD) and the RNA standards from the AccuBlue quantitation kit we see no material change in the amount of RNA present. This implies a lot of the RNA in the vaccines is in fact DNA-RNA hybrids which is resistance to DNaseI digestion.

This likely explains why many of the Moderna lots have very different amounts of Spike DNA contamination compared to Vector DNA. This is seen in Speicher et al Figure 5 where the red lines in many Moderna lots have many orders of magnitude more Spike DNA (Red) compared to Vector DNA (Blue) as assessed by qPCR. Their DNaseI conditions are doing a better job of eliminating the DNA that has no RNA complement. Not all of the Moderna lots exhibit this behavior and none of the Pfizer lots exhibit this behavior.

There are some DNases on the market that claim to do a better job digesting DNA in DNA:RNA hybrids.

One is known as DNaseI-XT from NEB

Its possible that Moderna and Pfizer are using different DNases in attempt to do a better job eliminating the DNA contamination and this is why we see differences in Spike/Vector DNA contamination in some lots.

Conclusions

Not only is there dsDNA contamination that exceeds the EMA and FDA limits in the modRNA vaccines, but we now know there is dsRNA contamination and RNA-DNA hybrids. What happens when this soup of contaminants is transfected into a cell is anyones guess.

We already know from the Kwon paper that cytosolic DNA transfections can trigger cGAS-STING and lead to oncogenesis.

dsRNA transfections will trigger the RNAi pathway.

DNA:RNA hybrid transfections are less studied.

Add in the complexity of the N1-methyl-Pseudo U modRNA and all the frame shifting it induces, and we have real bathtub gin masquerading as precision medicine.

We can only hope this doesn’t leave us Down with Disease…

with a fully tweaked Golgi Apparatus

Im off to the Sphere this weekend. First time in that venue. Wish me luck.