[LetThereBeLight]

The technology for synthesizing large quantities of specific RNA sequences has only been available recently. Same goes for forming the lipid nanoparticles that are used for encapsulating and delivering the mRNA. In fact if anyone has more detailed information on how these two processes are done I would love to learn more.

[rolph]

PCR, the Polymerase Chain Reation.

https://en.wikipedia.org/wiki/Polymerase_chain_reaction

the emphasis in the wiki article is on DNA, the same basic principles apply with RNA.

for example you can start with RNA and use reverse trancriptase to produce a DNA sequence, amplify that sequence to a large copy number by repeatedly replicating it then transcribe the DNA to produce large quantities of RNA.

or you can start with the DNA [in large quantities of purity] then create many copies of the RNA by repeatedly transcribing the DNA.

The practice of artificial [in vitro] gene synthesis can create arbitrary sequences for input to the process[es].

https://en.wikipedia.org/wiki/Artificial_gene_synthesis

liposomes are manufactured via biochemical-mechanical process.

https://en.wikipedia.org/wiki/Liposome#Manufacturing

[innomatics]

> the emphasis in the wiki article is on DNA, the same basic principles apply with RNA.

This is not my understanding. PCR works to produce DNA because the DNA polymerase enzyme creates DNA copies from DNA templates, and those copies become templates themselves, feeding back into the chain reaction.

I'm not aware of similar enzymes capable of making RNA copies from RNA templates (RNA polymerases use DNA templates AFAIK).

I was imagining the RNA vaccines would have used a fully synthetic oligo production method, to get the extreme purity required. But it might be that some sort of PCR-like amplification process is used. Would love to read more if someone has details.

[rolph]

the viral RNA polymerase replicates the viral RNA and this can be exploited for direct RNA to RNA amplification.

to wit : https://chempedia.info/info/viral_rna_polymerase/

I was making reference to the process of cyclic amplification, in a manner palatable to non biology literate individuals

The same _basic_ principles

as can be read in the chempedia example the process of RNApol replication of RNA from RNA template is error prone, thus it is superior to create a large copy number of DNA template corresponding to the mRNA desired as DNA amplification is self correcting for the most part.

dependent upon experimental or procedural requirements it may be desirable to produce error prone replication variants of the RNA template, however it is quite possible to go from RNA template to RNA product with RNApol.

due to the fragile nature of RNA there must be some shielding or , a low cycle rate is used in conjunction with immediate harvest and stabilization of the product.

the sars-2 covid19 virus replicates by way of an RNA dependent RNApol [rdRNApol]

https://pubmed.ncbi.nlm.nih.gov/32277040/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164392/

[innomatics]

Thanks for the additional links. I guess you would need temperature stable viral RNApol to do direct 'RNA' amplification. Not sure if that exists or if it's even possible due to RNA instability. As you say not so worthwhile due to high errors.

Producing RNA from DNA amplicons (PCR products) makes sense, I'm curious if that is the source of material from in the vaccines, or if it's artificially synthesized?

Would that be a trade secret?

[rolph]

the exact sequences could be proprietary secrets.

when you have determined a sequence of RNA corresponding to a desired protien you may construct a DNA template with modification to suit your purpose.

the DNA polymerase used for DNA PCR is originally from a high tempurature tolerant organism, [thermophillic bcterium] and it is possible via searching high and low to find an organism that bears high temperature tolerant rdRNApol

https://www.sciencedirect.com/topics/biochemistry-genetics-a...

and BTW the original inventor of PCR [kary mullis]

was awake AF on LSD when he conceived of this technology while sitting on a park bench.

https://en.wikipedia.org/wiki/Kary_Mullis

i believe the original PCR was performed MANUALLY! with glassware and perfectionist tedium.

https://en.wikipedia.org/wiki/Kary_Mullis#Use_of_hallucinoge...

you may find this to be interesting:

https://en.wikipedia.org/wiki/Ion_semiconductor_sequencing

[innomatics]

Thanks for the Kary Mullis story. I performed thousands of PCRs and even made some batches of taq many years ago but never knew about its ideation! I knew some pretty out there biochemists though.

[wrinklz]

I'd like to know more about the potential for lipid nanoparticles to cross the BBB [1], and therefore consideration of long-term effects of mRNA and resulting proteins in brain cells. Am I alone in this concern? [1] https://pubmed.ncbi.nlm.nih.gov/29886842/