DNA and RNA

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Tero
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DNA and RNA

Post by Tero » Tue Aug 18, 2020 11:20 am

I gave a class in the early spring on humans and DNA, and most of it went on the big scheme of things, taking the terms and connecting them to human populations, diseases etc.

I had really poor biology in college in the early 70s. I picked up a bit more at work after 1980. I worked with many biochemists, and did get Lehninger's Biochemistry at the time to see what we were working on. Generally I just had to work with papers on a single enzyme, so DNA was not important. But we did make some antivirals, so at least nucleosides were familiar. The little bits DNA is made of.

Just collecting a few concepts I have learned lately.

Sense and antisense. Only one strand has info for any particular section. You read from one "end" and there are start and stop codes that are easy to find.
Sense and antisense

A DNA sequence is called a "sense" sequence if it is the same as that of a messenger RNA copy that is translated into protein.[32] The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands can contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear.[33] One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.[34]

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes.[35] In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription,[36] while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.[37]
https://en.wikipedia.org/wiki/DNA#Sense_and_antisense

Gene mapping: from 5' to 3' /3' to 5'
https://biolympiads.com/principles-of-g ... -problems/
Last edited by Tero on Tue Aug 18, 2020 11:35 am, edited 1 time in total.
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Re: DNA and RNA

Post by Tero » Tue Aug 18, 2020 11:28 am

Viruses, bacteria etc can have double stranded DNA where there is info on both strands:
But keep in mind that sometimes, such as in prokaryotes, overlapping genes on opposite strands means the sense for one mRNA can be the antisense for another mRNA[2].
https://en.wikipedia.org/wiki/Sense_strand
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Re: DNA and RNA

Post by Tero » Tue Aug 18, 2020 11:37 am

A bit of confusion in the coding and template strands:
https://upload.wikimedia.org/wikipedia/ ... b/MRNA.svg
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Re: DNA and RNA

Post by Tero » Tue Aug 18, 2020 11:43 am

More on the confusion of sense and antisense. It seems to be a local convention, dealing with only the gene at hand:

Juan Pablo Matte Risopatron
Pontificia Universidad Católica de Chile
Sense and anti-sense is used as reference in regard to a specific gene. The name is used only locally in a segment of the whole DNA. For example an specific gene in the genome which coding region start from the 5’ to 3’, is called “sense”, the strand that goes in the “opposite” direction (which is also from 5’ to 3’) is called “anti-sense”. Moreover, genes are spread in the genome in both strand, and the direction of the genome is arbitrary to have a consensus of the localization of the genes. The important is that genes orientation is always the same (5’ to 3’), and in coding DNA, start codon (5’-ATG-3’) is located in the sense strand, where the complementary sequence is situated in the antisense strand (3’-TAC-5’). Nevertheless, the antisense strand is the one that is transcribed to give a sense mRNA with the start codon 5’-AUG-3’.

https://www.researchgate.net/post/If_I_ ... _antisense
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Re: DNA and RNA

Post by Tero » Wed Aug 19, 2020 1:16 am

Genes for most important proteins have been mapped. The documents and wikipedia draw a continuous chromosome with bands (these are from staining experiments). Insulin is found on chromosome 11 in the "reverse strand"
http://may2017.archive.ensembl.org/Homo ... 79-2161341
https://en.wikipedia.org/wiki/Insulin

the convention is to read DNA 5' top 3' end. This seems to indicate that you number the residues 5' to 3', but this protein is in the complementary band. To make it, you read again from 5' to 3', but now toward the "tip" of the chromosome.

Another example:
https://en.wikipedia.org/wiki/Angiotens ... ing_enzyme

http://may2017.archive.ensembl.org/Homo ... 1-63498380

this one is in the "forward strand"
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Re: DNA and RNA

Post by Tero » Sun Aug 30, 2020 2:12 pm

Imprinted genes: favoring either the gene from male or female parent

The majority of imprinted genes in mammals have been found to have roles in the control of embryonic growth and development, including development of the placenta.[23][47] Other imprinted genes are involved in post-natal development, with roles affecting suckling and metabolism.Another hypothesis proposed is that some imprinted genes act coadaptively to improve both fetal development and maternal provisioning for nutrition and care.[9][55][56] In it, a subset of paternally expressed genes are co-expressed in both the placenta and the mother's hypothalamus. This would come about through selective pressure from parent-infant coadaptation to improve infant survival. Paternally expressed 3 (PEG3) is a gene for which this hypothesis may apply.

23
https://www.sciencedirect.com/science/a ... via%3Dihub

wiki
https://en.wikipedia.org/wiki/Genomic_imprinting
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Re: DNA and RNA

Post by Tero » Sun Aug 30, 2020 2:44 pm

The imprinting example related to placenta in humans and horses:
https://news.cornell.edu/stories/2013/0 ... tudy-shows

DNA is silenced
https://en.m.wikipedia.org/wiki/Gene_silencing
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Re: DNA and RNA

Post by Tero » Wed Oct 14, 2020 5:39 pm

when you discover a tool in science, you publish it, it gets wide use immeadiately (socialism!) instead of patenting it (for a limited number of uses)
Then you get a Nobel
https://cen.acs.org/biological-chemistr ... mpaign=CEN
I think the video is playable to all
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Re: DNA and RNA

Post by Tero » Sat Dec 12, 2020 1:30 am

Not finding a lot on RNA production. It is inefficient chemically, so biological systems are used.
In Vitro Transcription
If you need long fragments of mRNA, we recommend RNA transcript synthesis. The yield of chemically synthesized RNA decreases exponentially with increasing length and is not suitable for synthetic long fragments of RNA. IntegrateRNA has developed a powerful T7 RNA polymerase-based transcription system that synthesizes up to 10 mg of mRNA in vitro in one day. RNA transcript synthesis is an alternative to chemical RNA synthesis and is very useful when long and/or modified capped mRNAs are required.
https://integraterna.creative-biogene.c ... gIW__D_BwE

This is the enzyme, but it needs some sort of cell to operate in
https://en.wikipedia.org/wiki/T7_RNA_polymerase

Vaccine paper pdf
https://stm.sciencemag.org/content/12/5 ... 96/tab-pdf
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Re: DNA and RNA

Post by Tero » Sat Dec 12, 2020 1:44 am

The methods section of that paper says BHK cells
https://en.wikipedia.org/wiki/Baby_hamster_kidney_cell

these are cultured cells, grown in feed and so on, harvested.
https://pubmed.ncbi.nlm.nih.gov/6218177/

The RNA is made by the cells, somehow harvested, processed into lipid packed particles.
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Re: DNA and RNA

Post by Tero » Fri Dec 25, 2020 5:50 pm

Tero wrote:
Fri Dec 25, 2020 1:40 pm
Pfizer vaccine. The DNA to RNA step still not clear to me. ecoli killed, no baby cells used.
Manufacturing
Pfizer is manufacturing the vaccine in its own facilities in a three-stage process. The first stage, conducted at a small pilot plant in St. Louis, involves the molecular cloning of DNA plasmids that code for the spike protein by infusing them into Escherichia coli bacteria. After four days of growth, the bacteria are killed and broken open, and the contents of their cells are purified over a week and a half to recover the desired DNA product. The DNA is stored in tiny bottles and frozen for shipment. Safely and quickly transporting the DNA at this stage is so important that Pfizer has used its company jet and helicopter to assist.[86]

The second stage is being conducted at plants in Andover, Massachusetts, and in Germany. The DNA is used as a template to build the desired mRNA strands. Once the mRNA has been created and purified, it is frozen in plastic bags about the size of a large shopping bag, of which each can hold up to 5 to 10 million doses. The bags are placed on special racks on trucks which take them to the next plant.[86]

The third stage is being conducted at plants in Kalamazoo, Michigan, and Puurs, Belgium. This stage involves combining the mRNA with lipid nanoparticles, then filling vials, boxing vials, and freezing them.[86] Croda International subsidiary Avanti Polar Lipids is providing the requisite lipids.[87] As of November 2020, the major bottleneck in the manufacturing process was combining mRNA with lipid nanoparticles.[86]
https://en.wikipedia.org/wiki/Tozinameran#Composition

ref 86
https://www.washingtonpost.com/health/2 ... facturing/

In Andover and Germany, scientists take the line of DNA and put it in an incubator with genetic building blocks to create messenger RNA.

The scientists then fill a special plastic bag with the purified messenger RNA and freeze it down. Each bag, about the size of a large shopping bag, contains enough for about 5 million to 10 million doses of vaccine. In Andover, the bag is hung on a special frame for handling and put on trucks to Kalamazoo.

The bags aren’t defrosted until everything is ready, because once they are, a clock starts ticking — the product has to be formulated and refrozen in under 72 hours, or it is wasted.

You don't just "make" RNA with DNA, you need cells or enzymes and RNA starting materials, nuclotides (triphosphates)
More
Manufacturing RNA-based biopharmaceuticals
As more experimental RNA drugs move through the clinic and into large-scale trials, the demand for efficient and cost-effective manufacturing strategies will grow (19). RNA-based biopharmaceuticals are inherently susceptible to endonucleases, so special handling is required for production and purification. Degradation of product during manufacturing adds heterogeneity and chemical instability to the product. Therefore, the manufacturing and purification methods used in RNA-based therapeutics differ from that of DNA and other proteins (20).

mRNA purification (post-chemical synthesis) includes concentration precipitation, extraction, and chromatographic methods (including high-performance liquid chromatography) (19). The purpose of the upstream concentration and diafiltration step is to concentrate (if lower titer) and change the buffer to the necessary pH and conductivity for the first chromatography step. The objective of the final concentration and diafiltration step is to de-salt and achieve the necessary final concentration prior to sterile filtration. A 5-kD membrane cut-off is generally used for concentration and diafiltration in mRNA processes. Because siRNA are smaller than mRNA, a 1-kD membrane cut-off is used for adequate retention of the siRNA product (21).
https://www.biopharminternational.com/v ... d-vaccines
20. T. Schlake et al., RNA Biol. 9 (11), pp. 1319–1330 (Nov. 1, 2012).
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Re: DNA and RNA

Post by Tero » Fri Dec 25, 2020 5:53 pm

Green Light, corporation, does cell free manufacture. You need to isolate enzymes. Some reagents are chemically made and introduced.
GreenLight takes a length of DNA that codes for the protein they want – the template. They put that into a vat full of free nucleotides, and enzymes will – just as in nature – start grabbing those nucleotides out of solution, making RNA copies of the DNA template.

The GreenLight system uses very simple nucleotides, which are made as byproducts of many biochemical processes and are often used in the production of baby formula.

GreenLight encourages them to react by adding a catalyst, an enzyme, just as in your cells. This reduces the amount of energy required to start the reaction.

You have to purify away all the things you used to make the messenger RNA by, in essence, filtering your material in a tube, which separates out the RNA molecules you want.

RNA molecules are delivered via lipid nanoparticles. Some lipid (fat) molecules have an interesting property: one end of each molecule is attracted to water molecules (hydrophilic), and the other end is repelled by them (hydrophobic).

When mixed with water and other molecules, such as RNA, the hydrophobic ends will huddle around the RNA, forming little spheres with the hydrophilic ends on the outside.
https://www.greenlightbiosciences.com/b ... a-vaccine/
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Re: DNA and RNA

Post by NineBerry » Sat Dec 26, 2020 11:43 pm

Really cool blogpost that goes deep into the details of how the Pfizer vaccine works. It's written for programmers.

https://berthub.eu/articles/posts/rever ... r-vaccine/

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Re: DNA and RNA

Post by Tero » Mon Dec 28, 2020 7:57 pm

DNA and RNA acting in early earth, without proper enzymes for support:
https://phys.org/news/2020-12-discovery ... -earth.amp
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Re: DNA and RNA

Post by Tero » Mon Dec 28, 2020 8:16 pm

NineBerry wrote:
Sat Dec 26, 2020 11:43 pm
Really cool blogpost that goes deep into the details of how the Pfizer vaccine works. It's written for programmers.

https://berthub.eu/articles/posts/rever ... r-vaccine/
Missing bit of structure on the right.
78CBDB65-F421-4DB2-BCCC-5545AC709C2C.jpeg
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