Tag: biotech

Chirality

Cosmic rays may explain life’s bias for right-handed DNA

Ultimately, the fact that researchers struggle to find a theory that balances the rise of chirality against the destruction of biological materials suggests that our ancestors may have been lucky to find that fine line. There is something special about planets like the Earth that protect this kind of chemistry.

2021-08-02: More details on this astounding achievement. They were able to sequence a 1.5 kilobase chiral DNA plus the Pasteur encoding stunt.

The chirally inverted L-DNA, possessing the same informational capacity but resistant to biodegradation, may serve as a robust, bioorthogonal information repository. Here we chemically synthesize a 90-kDa high-fidelity mirror-image Pfu DNA polymerase that enables accurate assembly of a kilobase-sized mirror-image gene. We use the polymerase to encode in L-DNA an 1860 paragraph by Louis Pasteur that first proposed a mirror-image world of biology. We realize chiral steganography by embedding a chimeric D-DNA/L-DNA key molecule in a D-DNA storage library, which conveys a false or secret message depending on the chirality of reading. Furthermore, we show that a trace amount of an L-DNA barcode preserved in water from a local pond remains amplifiable and sequenceable for 1 year, whereas a D-DNA barcode under the same conditions could not be amplified after 1 day.

2022-10-28: The same group doing the next step, a chiral RNA polymerase. This one is about 10% larger than previous work on the DNA polymerase as measured in kDA.

Zhu chemically synthesized a 100-kDA mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of high-quality l-RNAs as long as 2.9 kilobases. A massive, 883 amino acid protein, it lay well beyond the limits of traditional chemical synthesis. But an analysis of T7s x-ray crystal structure showed the enzyme could likely be split into 3 sections, each stitched from short segments. In solution, the fragments naturally folded into their proper 3D shapes and assembled themselves into a working T7.

The mirror-image RNAs fashioned by the polymerase were far more stable than the normal versions produced by a regular T7, because they were untouched by the naturally occurring RNA chewing enzymes that almost unavoidably contaminate such experiments and quickly destroy normal RNAs.

Now, Zhu needs to make the remaining components of a mirror-image ribosome. The 3 RNA fragments they synthesized make up 66% of the total mass of a ribosome. What remains are the 54 ribosomal proteins and several proteins that work in concert with the ribosome, all of which are smaller and thus likely easier to synthesize. Then the question is whether the full parts kit will assemble into a ribosome.

Even if they do, the resulting molecular machines might still not be functional. In order to churn out proteins, ribosomes must work in conjunction with a suite of additional helper proteins. To make this work inside a living cell, Church thinks it will be necessary to rewrite an organism’s genetic code so the engineered ribosome can recognize all those proteins, particularly the 20 that ferry amino acids for building new proteins.


2023-08-28: nonlinear optics to detect chirality

They have realized a technique that can completely distinguish enantiomers in solution in an all-optical manner: no chemical tags, no particular UV/VIS absorbance needed from the compound structures, etc. And it is extremely fast and extremely sensitive, as opposed to traditional methods like optical rotation, circular dichroism, etc.

2023-09-08: What about magnetism?

Magnetic surfaces on minerals in bodies of water on the primordial Earth, charged by the planet’s magnetic field, could have served as “chiral agents” that attracted some forms of molecules more than others, kicking off a process that amplified the chirality of biological molecules, from RNA precursors all the way to proteins and beyond. Their proposed mechanism would explain how a bias in the makeup of certain molecules could have cascaded outward to create a vast network of chiral chemistry supporting life.

It’s not the only plausible hypothesis, but “it’s one of the coolest because it ties geophysics to geochemistry, to prebiotic chemistry, and ultimately to biochemistry”

Biosensor scaling

The tests are performing well, but are still being optimized. The biggest hold back at the moment is to scale from mass production (10s of 1000s of chips per month) to extreme mass production (10s of millions and, with time, 100s of millions of chips per month).

2021-10-07: This seems to have happened:

Covid-10 has driven a change in diagnostics technology. Many companies debuted new technologies for at-home test kits under the FDA emergency use authorization last year. “It’s going to go beyond Covid-19 testing. Everything from cancer detection to STD testing and anything that would go through the historical lab chain. We’ve gotten a lot of inquiries for rapid test kits from various companies about new applications. It’s going to be a revolution for the diagnostics market. To go from a test that would take 24-48 hours and cost $150 to get processed, to something you can do in 15 minutes for $20 or $30.”

Glycans

There’s a reason why genomics and proteomics have leapt ahead of glycomics: The sheer complexity of sugars makes them more difficult to study. DNA, RNA and proteins are linear molecules built according to defined sets of rules, and scientists have the tools to sequence, analyze and manipulate them. But glycans are branching structures that assemble without a known template. The same site on 2 identical proteins might be occupied by very different glycans, for instance. Glycans also have exponentially more potential configurations than DNA or proteins: 3 different nucleotides can make 6 distinct DNA sequences; 3 amino acids can make 6 unique peptides; 3 glycan building blocks can form more than 1000 structures. Glycans are flexible, wobbly and variable; intricate, dynamic and somewhat unpredictable. Their analysis demands greater technical expertise and more sophisticated equipment.

Where Do New Genes Come From?

some “orphan genes” with no obvious ancestors evolve out of junk DNA, contrary to old assumptions.

see also this older article

several genes that were present in only 1 or 2 species and not others, suggesting that these genes weren’t the progeny of existing ancestors. Begun proposed instead that random sequences of junk DNA in the fruit fly genome could mutate into functioning genes.

Noncanonical ORF

Using mass spectrometry, ribosome profiling, and several CRISPR-based screens, Chen et al. identified 100s of previously uncharacterized functional micropeptides in the human genome. Protein translation outside of annotated open reading frames (ORFs) in messenger RNAs and within ORFs in long noncoding RNAs is pervasive. A functional screen using CRISPR-Cas9 with single-cell transcriptomics suggested critical roles for 100s of micropeptides. Micropeptides encoded by multiple short, upstream ORFs form stable protein complexes with the downstream canonical proteins encoded on the same messenger RNAs. One of the particularly puzzling things is that we’re seeing proteins that have real functions but do not seem to be evolutionarily conserved. Noncoding RNA may actually be coding.

Biotech reproducibility

DARPA made it a condition of funding to pair research teams in biotech with independent verifiers, and learned a ton. They talk about bringing more engineering to biosciences. Very encouraging, I hope this catches on.
2023-04-04: The number of problems reproducing mouse studies is daunting. Perhaps more organelles can be part of the solution.

  • The Bible of mouse caretaking is called the Guide for Care and Use of Laboratory Animals. It did not change at all between 1972 and 2019, despite everything I’m about to tell you.
  • Mice raised at 5 different animal facilities in Europe, under otherwise identical conditions, have “persistent differences in body weight” and behavior. There are even differences in how their genomic DNA is packaged inside of neurons. Nobody really knows why.
  • If 2 different scientists at the same university carry out the same experiment on mice, their results will be more replicable than the same experiment, carried out by the same person, at separate universities.
  • Mice that give birth in cages with little toys or knick-knacks produce more pups. Those pups are larger after 21 days.
  • Smaller cages cause *some* strains of male mice to fight more often. (There’s an entire meta-analysis on this topic alone, which I’m sure you’d find riveting.)
  • “Mice housed on deep bedding had smaller adrenal, kidney, liver and heart weights as well as larger body and tail lengths compared with groups kept on shallow bedding” after just 12 weeks.
  • Mice handled by male scientists feel less pain. The finding holds true when a female scientist does the experiment but holds a t-shirt, previously worn by a man, near the mouse. The effect fades away after 30 minutes.
  • Mice spend less time licking an irritated part of their body when a human is nearby, “even if that ‘person’ is a cardboard cutout of Paris Hilton.”
  • Animals stored on higher shelves are more stressed and have impaired immune systems, probably because these areas are closer to lights and vibrate more.
  • Mice exposed to even dim light during the night (e.g. an LED on a computer monitor) “had a body mass gain…50% more than other mice that lived in a standard light-dark cycle.”
  • After just 4 weeks, mice exposed to a dim light during the night ate more than those in complete darkness. (Mice, like humans, raid the proverbial refrigerator when they can’t sleep.) Many genes linked to inflammation were also activated.
  • Mice kept in cages with wood chip bedding eat 1.5 grams of their mattress every day. This changes the bacteria in their microbiomes.
  • Historically, male mice were used in research 6x more often than females because they don’t have an estrous cycle, and their metabolisms were therefore thought to be more predictable. About 80% of drugs are tested only on male mice. Some drugs, notably Ambien, are more potent in females and cause more side effects.
  • Female mice are less erratic than males while exploring an open space. The estrous cycle of female mice shows only “a very weak effect on their behavior.” Other scientists have said much the same thing since at least 2018.
  • The body temperature and activity patterns of male mice vary more in “a day than females do across an entire estrous cycle.”
  • 6% of all mouse genes are regulated in sex-specific ways. The expression level of 1k genes varies between males and females, and the level of another 600 genes wobble, up and down, during a female’s estrous cycle.
    • The genes turned on in a specific type of immune cell, called a neutrophil, also differ between males and females.
  • The genes associated with longer lifespans in mice differ between males and females, based on data from “3k genetically diverse animals raised in tightly-controlled, homogenous conditions.”
  • Grain-based food usually contains unknown amounts of phytoestrogens, which change the onset of an animal’s puberty.
  • The standard diet for mice, called AIN-93, hasn’t changed in 30 years. But manufacturing of that food does: Even if you use “the same grain-based diet used in the past by others, its composition will likely differ.”
  • The sexes of a mouse’s siblings can skew an animal’s behavior. “Female-skewed litters demonstrated more social play, while the male-skewed litters demonstrated more solitary play.”
  • Rooms with a higher humidity are associated with lower pain thresholds for mice scorched with hot water. Nobody really knows why.
  • Most animal facilities change the bedding for mice every week, which causes “heart rate, blood pressure, and locomotion in both male and female mice” to spike for between 75 – 105 minutes. This effect persists even after mice have been moved many times.
  • Mice exposed to a regular, 37 Hertz magnetic field spend less time exploring open spaces, and more time sleeping.
  • Mice are kept in rooms between 21 and 26 degrees Celsius. “But the natural comfortable temperature for mice is warmer — between 30 and 32 degrees Celsius.” Colder mice experience more stress, their tumors grow faster than mice kept in warm rooms, and “mice genetically modified to develop obesity only gained a lot of weight at warmer temperatures but not at colder temperatures.”
  • Keeping mice at higher temperatures also blunts their muscle gain after exercise.
  • 2 mice of the same strain are often genetically distinct, even if they’re labeled as isogenic. DNA differences accumulate over generations of breeding.
  • Sometimes, scientists use the entirely wrong mice, or swap 2 mouse strains accidentally, and have to retract their papers afterward.
  • The microbiome of an animal can influence the effects of a drug. In 1 instance, a lab at Michigan State University was “testing how a certain drug affects bone density, and they found that treated lab mice lost bone compared with controls.” When they repeated the experiment on identical mice, of the same strain and from the same vendor, those mice gained bone density. A 3rd experiment found no effect. Each animal had a different microbiome.

50% crazy

When I invest in biotech, I have a sort of a model for the type of person I’m looking to invest in. There’s sort of a bimodal distribution of scientists. You basically have people who are extremely conventional and will do experiments that will succeed but will not mean anything. These will not actually translate into anything significant, and you can tell that it is just a very incremental experiment. Then you have your various people who are crazy and want to do things that are going to make a very big difference. They’re, generally speaking, too crazy for anything to ever work. You want to find the people who are roughly halfway in between. There are fewer of those people because of these institutional structures and whatnot, but I don’t think they’re nonexistent. My challenge to biotech venture capitalists is to find some of those people who are crazy enough to try something bold, but not so crazy that it’s going to be this mutation where they do 100 things differently.