Immunity by incompatibility – hope in chiral life

[Duda Jarek]

There are programs searching for minimal cell (e.g. https://www.nature.com/articles/s41586-023-06288-x ) but for full synthesis it is not needed.

Being able to synthesize mirror ribosome and DNA/RNA, they could get all required enzymes - then enclose it into a membrane ... and why wouldn't such minimal cell work? (to be further extended with more functions)

From fig 2.4 of the report:

This is bottom-up approach, but the report also discusses top-down: trying to stepwise convert natural cell into mirror one through various approaches, e.g. genetic code reprogramming like replacing tRNA with carrying mirror amino acids.

Section 2.3 of report:

"1. Production of mirror-image proteins in vivo by creating a crossover pathway made of natural-chirality components.
2. Production of mirror-image proteins in vivo by creating an entirely mirror-image central dogma.

3. Delivery or assembly of a full mirror-image DNA genome in vivo, and removal of the natural-chirality genome, to create a mirror bacterium."

 

Edited Tuesday at 06:57 AM by Duda Jarek

[CharonY]

9 hours ago, Duda Jarek said:

Being able to synthesize mirror ribosome and DNA/RNA, they could get all required enzymes - then enclose it into a membrane ... and why wouldn't such minimal cell work? (to be further extended with more functions)

It simply doesn't. What we know is that the a lot of more is needed. There is a list of things folks assume is needed, but so far putting them into a membrane has not yielded a viable cells. This is why I mentioned that we need to figure out what is needed minimally first, as obviously we are still missing critical elements.

Again, what you proposed is early thinking about cells and as it turns out again and again, it does not result in viable cell. That is why with enormous financial investment at that time, the only thing folks were able to come up with was to remove DNA and then put a reduced version back into the cell it came from. The graph is basically ignoring all the critical steps. It is a bit like:

Build rocket-> develop system for FTL-> explore different star systems.

Also, while the authors acknowledge that those very theoretical organisms would need to compete with existing organisms for molecules with the more common chirality, they actually just speculate that they will somehow overcome that. This suggest that you would need to bioengineer all the contingencies into the system, which normal bacteria are able to do from the get go.

The authors are skipping a lot of steps, and from my perspective, these steps are the actual challenges.

[Duda Jarek]

Yes, they use this vague "boot" verb for kind of bringing life to a synthetic cell - but what exactly is it?

Naively there is just chemistry - statistical reactions in cytosol as a dense soup of biomolecules ... recreating composition of this soup (for some minimal cell with just central dogma), what more is needed to make it alive?

 

[CharonY]

2 minutes ago, Duda Jarek said:

Yes, they use this vague "boot" verb for kind of bringing life to a synthetic cell - but what exactly is it?

Naively there is just chemistry - statistical reactions in cytosol as a dense soup of biomolecules ... recreating composition of this soup (for some minimal cell with just central dogma), what more is needed to make it alive?

 

 

That is the exact issue/question: what is needed to make it alive? We don't know and have not been able to achieve it. It is the same as FTL- we merely have to find a way to overcome the speed of light limit. Naively, it is just bending time and space. How hard can that be?

Again, until someone actually figures out how to build a cell from scratch, we are talking hypotheticals here. Not that this might not be important at some point in the future, but in contrast to many other things that are a risk right now, it is still a hypothetical. Honestly, if we wanted to prevent us dying from infections, someone should figure out how to restore trusts in vaccinations and go from there. Mirror or not, organisms will have a 3d structure to work on.

[Duda Jarek]

While as a physicists I can say FTL is forbidden by special relativity (but in theory allowed by general relativity) ... I honestly cannot imagine obstacles for recreating cell chemistry - generally easily made outside cell ... so recreating original cytosol composition of some minimal cell, why wouldn't it work? Which chemical processes? They would work individually, but couldn't synchronize like in a living cell?

Anyway, looks like these are your words against their. Searching for "boot synthetic cell" I see lots of positive examples. Could you support your view with some references?

ps. Lots of talks from Build-a-Cell seminar (tomorrow about mirror bacteria I plan to visit): https://www.youtube.com/playlist?list=PLb2LmjoxZO-gKWXZZadcko8tHHkPuEeJT

E.g. 2020 John Glass from J. Craig Venter Institute (e.g. minimal genome: 483 kbp, 432 proteins, 39 RNAs):

 

Edited yesterday at 04:09 AM by Duda Jarek

[Duda Jarek]

Just watching a week old presented by Yutetsu Kuruma from Japan Agency for Marine-Earth Science and Technology: Design and construction of artificial cells based on cell-free system - mentions the most difficult is cell replication, where I completely agree ... but just recreating (mirror) central dogma looks doable (?) - and might be sufficient for safe mass production of mirror biomolecules (?) ... or will become the first step toward replicating mirror cells ...

George Church Synthetic genomes & tRNAs in vitro & vivo (Nov 2024):

 

Edited yesterday at 05:42 AM by Duda Jarek

[CharonY]

12 hours ago, Duda Jarek said:

mentions the most difficult is cell replication, where I completely agree ... but just recreating (mirror) central dogma looks doable (?) - and might be sufficient for safe mass production of mirror biomolecules (?) ... or will become the first step toward replicating mirror cells ...

Producing chiral molecules have been done for over a decade at least (perhaps longer), so that is nothing new. The central dogma sounds pompous, but is really just transcription/translation and can be easily done in vitro without cells for a while, too. This is not what is tricky. Replication, sustainable metabolism and overall maintaining cellular integrity, are the tricky bits (plus a few I am missing, I am sure).

[Duda Jarek]

Sure a company could now produce mirror protein drug for maybe thousans of customers ... but what if millions would like to buy it?

If we agree synthetic central dogma is doable, why they couldn't build on it adding further features? Where exactly is your boundary you believe they will not be able to cross?

[CharonY]

45 minutes ago, Duda Jarek said:

Sure a company could now produce mirror protein drug for maybe thousans of customers ... but what if millions would like to buy it?

I am not sure what you think the issue is. There are plenty of drugs that are chiral often only or the other is used (or sometimes mixtures of both).

Are you also worried about naturally occurring mirror molecules?

[Duda Jarek]

Sure one can synthesize proteins from single amino acids, but comparing with its production by bacteria, what would be the difference in cost and volume? A thousand? A million?

My point is that there will be large financial incentives for such cost reductions and scaling up, and there is a quickly growing number of potential applications, e.g. from the report:

Quote

Box 2.1: Applications of mirror peptides and proteins
Peptides are promising therapeutics due to their ability to bind drug targets with high affinity. However, natural-chirality peptides suffer from comparatively low biostability as they can be degraded by proteases (enzymes that degrade peptides and proteins) that are abundant in most living organisms and play important roles in the immune system (Muttenthaler et al., 2021). Scientists are therefore exploring the use of additional building blocks, including ᴅ-amino acids, to modify peptides in order to create biostable peptide-like drugs (Muttenthaler et al., 2021). In a classical approach, a peptide made of natural-chirality ʟ-amino acids is first developed using standard drug discovery techniques, and then modified to include non-canonical building blocks, including ᴅ-amino acids, to
improve its stability while retaining its biological activity (Imanishi et al., 2021; Muttenthaler et al., 2021). For example, etelcalcetide, a drug for the treatment of secondary hyperparathyroidism, consists of a 7-amino acid ᴅ-peptide linked to a single ʟ-amino acid (Blair, 2016).

Mirror-image proteins might also be used in drug discovery to aid the development of stable fully mirrored peptide drug candidates. Scientists can use standard drug discovery techniques to find natural-chirality peptides that bind to mirror-image versions of a drug target. By chiral symmetry, the mirror-image version of the peptide will then bind to the natural-chirality version of the target in patients, but is expected to have a significantly improved half-life because it would be resistant to protease degradation and immune recognition (Callahan et al., 2024; Harrison et al., 2023; Muttenthaler et al., 2021; Welch et al., 2010).

Several other applications of mirror-image proteins have been explored to date. For example, mirror-image proteins have been used to enable protein structure determination by X-ray crystallography. Chiral proteins can only form crystals in chiral space groups, whereas racemic mixtures can form centrosymmetric crystals, which significantly simplifies phase determination and thereby aids resolution of the protein structure (Mackay, 1989). This approach has been successfully used to determine a number of protein structures (Avital-Shmilovici et al., 2013; Banigan et al., 2010; Dang et al., 2016; Harrison et al., 2023; Huang et al., 2016; Hung et al., 1999; Luisier et al., 2010; Mandal, Pentelute, Tereshko, Kossiakoff et al., 2009; Mandal, Pentelute, Tereshko, Thammavongsa et al.,
2009; Payne et al., 2021; Pentelute et al., 2010; Teng et al., 2021; Yeung et al., 2016).

Quote

Box 2.2: Applications of mirror nucleic acids
DNA is commonly synthesized for recombinant DNA technology, but nucleic acids have numerous additional applications in basic science, biotechnology, and medicine. A drawback of DNA and RNA in these applications is their limited biostability, as they are recognized and degraded by biological processes. Mirror DNA and RNA retain properties of DNA and RNA, including the ability to form predictable structures determined by base-pairing, but are more stable as they are not susceptible to degradation by nucleases (enzymes which break down DNA and RNA) which are highly abundant in the environment.

These results led to interest in using mirror oligonucleotides as aptamer therapeutics and in other applications. Aptamers are oligonucleotides that can bind with high affinity to some biological molecule, e.g., a drug target. Aptamers can be discovered rapidly through directed evolution (Tuerk & Gold, 1990). However, similar to natural-chirality peptides, natural-chirality oligonucleotides are generally too unstable in physiological environments
to be useful as aptamer drugs. In contrast, mirror-image aptamers—termed Spiegelmers by TME Pharma, the company developing them—could retain the binding properties but exhibit greater biostability and thus become useful drugs. Two Spiegelmers have reached early clinical testing as of 2024 (Kaur et al., 2018).

Mirror oligonucleotides might have diverse applications beyond aptamer therapeutics. For example, molecular beacon or riboswitch nucleic acids have been explored as biosensors (Seeman & Sleiman, 2017), and mirror-image versions of these can recapitulate their key properties while being more stable. Mirror image DNA nanostructures are being explored as drug delivery systems due to their greater resistance to serum host nucleases (Kim et al.,
2016). They could also serve as interesting materials in DNA nanotechnology due to their ability to form predictable shapes with other mirror nucleic acids without interfering with natural-chirality nucleic acid assemblies (C. Lin et al., 2009).

> Are you also worried about naturally occurring mirror molecules?

For naturally appearing around us, evolution should generally prepare us for.

But for others it did not, what does not automatically mean toxicity, but that there is a probability of various toxicities due to looking random interactions (again thalidomide example) ... and the number of potential interactions grows with the square of number of chiral biomolecules, so multiply this probability by millions e.g. for necrosis of mirror bacteria in human bloodstream - for me it sounds worrying.

[CharonY]

1 hour ago, Duda Jarek said:

For naturally appearing around us, evolution should generally prepare us for.

Then how about the many unnatural drugs? Are you worried about those, too? Because that argument applies to to any newly synthesized compound and materials.

[KJW]

1 hour ago, Duda Jarek said:

one can synthesize proteins from single amino acids

What's the largest protein molecule that currently can be synthesised by purely chemical means?

Does the technology exist to synthesise proteins in vitro from mRNA using purified ribosomes and the tRNAs? I'm aware that attaching an alternative amino acid to a tRNA molecule will place that alternative amino acid into the growing polypeptide, so that one could synthesise mirror proteins from the mirror amino acids alone (using natural mRNA, ribosomes, and tRNAs).

 

[CharonY]

53 minutes ago, KJW said:

Does the technology exist to synthesise proteins in vitro from mRNA using purified ribosomes and the tRNAs? I'm aware that attaching an alternative amino acid to a tRNA molecule will place that alternative amino acid into the growing polypeptide, so that one could synthesise mirror proteins from the mirror amino acids alone (using natural mRNA, ribosomes, and tRNAs).

Yes, we can do in vitro protein expression, fairly easily and routinely. D-proteins are mostly used in structural investigations (I am not sure whether any with therapeutic value have been discovered yet). However, smaller peptides including either some or consisting of D-amino acids are either in use or being developed. DADLE is a synthetic peptide that has been synthesized in the 90s, for example.

 

Edit to add:

In isolation, mirror protein, amino acids, DNA and so on are not particularly more dangerous than any other drug or synthesized compound. The risk of mirror organism is entirely independent of that, and hinges on the ability on creating that in the first place. Just adding some chirality does not add much. Bacteria routinely use many tricks, such as sugars in many shapes and forms to confuse our immune system. In fact, in their O-antigens one can find D- and L-forms of their subunits to confuse our immune system. I.e., this is not fundamentally new chemistry we are talking about here.

[KJW]

On 12/16/2024 at 9:03 PM, Duda Jarek said:

This is entire Chapter 4: Risks to Human Health of https://purl.stanford.edu/cv716pj4036, maybe take a look there.

For example for macrophages looks like nearly nothing would work:

Considering the chemotaxis, adherence, and internalization steps above, it is my understanding that foreign bodies not directly recognised by macrophages can be tagged by opsonins so that they become recognised by macrophages. Thus, it would appear that mirror bacteria can be killed by the immune system even if the process is less efficient.

 

[Duda Jarek]

6 hours ago, CharonY said:

Then how about the many unnatural drugs? Are you worried about those, too? Because that argument applies to to any newly synthesized compound and materials.

I have some experience in chemoinformatics (e.g. https://link.springer.com/article/10.1007/s11030-022-10589-0 predicting probability distributions of ADMET properties like cardiotoxicity) and yes - excluding various toxicities is extremely crucial and difficult part of drug design, from virtual screening to clinical trials.

4 hours ago, KJW said:

Considering the chemotaxis, adherence, and internalization steps above, it is my understanding that foreign bodies not directly recognised by macrophages can be tagged by opsonins so that they become recognised by macrophages. Thus, it would appear that mirror bacteria can be killed by the immune system even if the process is less efficient.

Some mechanisms should still work (e.g. achiral) and Chapter 5: Medical Countermeasures of the report discusses some approaches like achiral antibiotics, producing mirror antibiotics, or releasing mirror bacteriophages ... but many would not - as in immunodeficient persons we should compare with.

Quote

Even if existing antibiotics or novel antimirror compounds could kill mirror bacteria, they would not necessarily be clinically effective. As discussed in Chapter 4, human immunity is anticipated to be impaired versus mirror bacterial challenge, and infection is frequently life-threatening in roughly analogous immunodeficiencies despite available options for antibiotic treatment. For antimirror compounds to be useful therapeutically, infections would need to be diagnosed and treatment begun before irreversible harm had occurred. It is therefore not clear that post-infection antibiotics would be sufficient to cure a mirror bacterial infection, or to mitigate serious health effects even if they could prevent fatality.

From the other side, mirror bacteria would indeed have crippled mechanisms like adhesion, also feeding with natural components - but there are also many achiral sources (Table 1.1: Achiral organic molecules that can be utilized by wild-type or mutant E. coli K-12), and bacteria can adapt to feed on mirror sugars ... the question is if it can get into the bloodstream, e.g. through injuries, if so it could rather easily exponentially grow in population leading to e.g. sepsis for example from released mirror proteins in necrosis.

And getting mirror bacteria, it would evolve, take new ecological niches having practically no natural enemies ... also malicious players could easily modify them to become more dangerous.