John Baross of the University of Washington and Gary Ruvkun of Harvard Medical School were the moderators for the session on life detection techniques: Where we are today and what to look for, in terms of life as we know it and life as we don’t know it?
Ben Clark of the Space Science Institute started his talk with an image of a Viking lander with Carl Sagan beside it. The Viking landers were the first and last spacecraft to be subjected to dry heat sterilization. Both were heated to a temperature higher than boiling water. Heat sterilization can now be done cheaper today, which might be necessary for future astrobiological missions. A third Viking lander had been completely built with flight hardware, but eventually, the proposed follow-on mission, which included tracks in place of footpads to transform it into a rover, was dropped because of the high costs of developing the Space Shuttle Program.
The Viking missions had a gas chromatograph–mass spectrometer (GCMS) to look for organics using hydrogen carrier gas. Initially having four planned biological experiments on Viking, it was reduced to three after serious budget overruns on the overall instrument. The final three selected were very general and simple experiments: pyrolytic release, labeled release, and gas exchange. Despite the tests being simple to perform in the laboratory, they were difficult to implement: all three were packed into a space of about 1 cubic foot. Due to the engineering challenges, Clark said that it was the most expensive instrument that had ever been developed for spaceflight.
The pyrolytic release exposed the soil to a solar simulator light source and radiocarbon monoxide and dioxide and looked for incorporation into complex organics, which would indicate the presence of life. The labeled release provided some common organic substrates used by biology and looked for the conversion of these to any kind of gas, such as CO2. The gas exchange used what was called “chicken soup,” a mix of everything that they thought life might need or be able to utilize. It then looked for changes in gas concentration. Each experiment had different modes to test in, which together spanned dry, moist, and wet conditions.
The pyrolytic release experiment, Clark said, had an overall negative result for life, although there was one data point that was anomalously positive. The labeled release experiment, on the other hand, did get indications of a
positive result. The result for the gas exchange experiment was negative, but a release of oxygen after humidifying the soil was observed, which indicated the presence of oxidants—a big surprise to the team. It also raised major doubts as to whether the labeled release results were just oxidants undergoing inorganic chemical reactions or if the result was truly of biological origin. An abiological way to simulate those results using oxidants was quickly discovered.1 The GCMS gas-exchange experiment found no organic molecules with an upper limit of <1 parts per billion (ppb).
Clark said that there was and still is some uncertainty about what was actually found on Mars with the Viking landers. There is the one anomalous data point in the pyrolytic release experiment. In the labeled release experiment, the injection of nutrients in an aqueous solution onto the soil began a rapid evolution of the radioactive gas, which then plateaued as if the nutrient were being used up.2 This was compared against a control sample of soil that was baked at 160°C for 3 hours, which yielded a null result. When they injected more aqueous nutrients into the soil about 8 days after the initial injection, they would have expected to see life metabolize that injection as well. However, the signal counterintuitively dropped (see Figure 4.1). This divided the community into two camps: those who thought the experiment showed biological activity and those who thought it was all abiotic. The abiotic camp thought that the first injection may have just been oxidants in the soil that were oxidizing the nutrients, specifically formate, while the biological camp said it was life metabolizing it. In the second injection, the abiotic camp said that the oxidants were consumed in the first injection, while the biological camp thought that the liquid was being chemically re-absorbed and that the organisms had died or become inactive. Oscillations in the data were attributed to uptake by minerals in the wet soil due to temperature variations by the abiotic side, while the biological side suggested it could be a circadian rhythm. The control sample, which was heated to 160°C for 3 hours, was used as proof by the biological camp, but the abiotic side said that the oxidants could be heat labile, that
1 C. Ponnamperuma, A. Shimoyama, M. Yamada, T. Hobo, and R. Pal, 1977, Possible surface reactions on Mars: Implications for Viking biology results, Science 197:455.
2 G. Levin and P.A. Straat, 1976, Viking labeled release biology experiment: Interim results, Science 194:1322-1328.
there could be multiple oxidants, or that the water released by heating the control sample could have destroyed the oxidants. The Curiosity rover, Clark noted, has also seen significant water release from samples heated to 160°C.
Clark said that two of Viking’s major accomplishments were that it was the first in situ, robotic mission looking for biomarkers on an extraterrestrial body and that it was also the first search for metabolic activity. No subsequent search for metabolic activity has yet been attempted. Because there are oxidants in the soil, any organics could be reacting with them during the pyrolytic step used to volatilize organics. Curiosity’s Sample Analysis at Mars (SAM) instrument is making corrections for this. The best idea to avoid this problem, according to Clark, is to use laser desorption mass spectroscopy, which will fly on the ExoMars rover mission (launch date 2020), led by the European Space Agency (ESA) and the Russian space agency, Roscosmos.
On the labeled release results, Clark said that several people tried simulating the results with different oxidants in the soils and achieved varying degrees of reproducibility. The most successful result so far, according to Clark, has been Quinn et al. (2013).3 Their experiment used perchlorates, which could not be measured by Viking but were discovered by the Phoenix mission. When irradiated by simulated cosmic rays, perchlorates transform into hypochlorite and trapped oxygen, which can then mimic the Viking results.
There were several things the Viking missions did not explore either. They did not provide all the possible metabolites in their biological experiments, such as H2, H2S, NO, or NO2. They analyzed only soil, and they did not test inside rocks or salts. The GCMS could not detect a biomass density of <1,000 microbes/cm3. Clark thinks that only (cold) sample return will let us determine whether or not martian material contains life or the signs of extinct life. The third, unused Viking lander, he said, was also considered for use in a sample return mission. Sample return is more feasible nowadays. However, as Clark said, the saying in their community is that a “Mars sample return is always 10 years away,” consistent with the 2026 launch date for the sample return envisioned by Dr. Stofan in a previous talk (see Chapter 2).
Clark then noted that many comments have been made lamenting that the Viking missions were not able to benefit from today’s knowledge of Mars during its mission design phase. However, Clark said that there actually was much known about Mars at the time and that some of the experiments would still be valid today. He then used the National Research Council report Biology and the Exploration of Mars4 (1966) to show what was already known about Mars, such as its incident ultraviolet (UV) flux, low temperature, and dry air, as well as the existence of extremophile organisms on Earth. Clark then cited a statement in the report that a negative result for organic molecules would preclude the existence of biology, which presumably biased the entire endeavor because no organics were measured by the GCMS.
Clark then said that Viking taught us new things about the martian soil. For one, they found high levels of iron in the soil. That in itself was not surprising, since Mars is red. However, they also found that levels of sulfur (in sulfates) and chlorine (in chlorides) were approximately 100 times higher than would normally be expected for soil on Earth, Mars, or the Moon. The Phoenix lander also found the oxidants perchlorate and chlorate in addition to the chlorides. It was also discovered that the soil at the two Viking sites, on opposite sides of the planets, were virtually identical. He then said that there is so much sulfur and chlorine salt in the soils that if you take a regolith, fill the porous space with ice (which happens on Mars), and then melt it, the result would be salt concentrations at Dead Sea levels. The magnesium sulfate salt is different from Earth’s NaCl salt, but it still has a high ionic strength. This would require organisms to have a high salt tolerance. Clark is collaborating with biologists who have taken salt-tolerant organisms and exposed them to perchlorates. They have found that alkali perchlorates are tolerated better than alkaline earth perchlorates. However, Mars seems to have alkaline earth magnesium and calcium perchlorates. This might not be relevant though, considering terrestrial microbes have no evolutionary reason to tolerate perchlorates.
Possible Life on Mars Today
Clark then switched to whether or not they now think that Mars has extant life. He said that, with the information they now have, they think it’s even more likely than they originally thought back during the Viking mis-
3 R.C. Quinn, H.F.H. Martucci, S.R. Miller, C.E. Bryson, F.J. Grunthaner, and P.J. Grunthaner, 2013, Perchlorate radiolysis on Mars and the origin of martian soil reactivity, Astrobiology 13:515.
4 National Research Council, Biology and the Exploration of Mars, National Academy Press, Washington, D.C., 1966.
sions. One bit of new information is that near-surface ice has been discovered at the Phoenix landing site. It was also discovered that Mars has a wide range of obliquity (tilt of the spin axis with respect to orbital plane), which allows for cyclic climate change. It is now in a low obliquity era, meaning that it is in a cold spell. The team actually saw frost in the wintertime at the Viking 2 landing site, which was a surprise. The Shallow Subsurface Radar (SHARAD) instrument on NASA’s Mars Reconnaissance Orbiter (MRO) recently discovered a huge quantity of shallow permafrost ice in the Utopia Planitia region where Viking 2 landed.
Conditions on Mars during the Noachian era (~3.7 to 4.1 Gyr ago) were favorable for the origin of life, according to Clark. It had liquid water and a dense atmosphere. Mars also apparently had a significant amount of H2, and possibly CO and CH4, in the atmosphere to provide energy for life. He then referred to a claim that 99 percent of organisms can metabolically use H2. Sulfur, which is found everywhere on Mars in the form of sulfate, could also have been a source of energy for chemoautotrophic life by combining with H2. It is even possible to have photosynthesis using sulfur-bearing instead of oxygen-bearing molecules. Using H2S instead of H2O was actually the first form of photosynthesis on Earth, he said. Mars may also have had organic molecules. Additionally, the planet received enough sunlight (43 percent that of Earth’s) to easily allow for photosynthesis. Both iron and manganese can be used as electron acceptors, and both are found on Mars. More broadly, Mars has all the CHNOPS (carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur) elements plus other key elements (Fe, S, Ni, Zn, Cu, Mn, and P) necessary for life at concentration levels, which suggest prior interactions with liquid water. Hydrothermal activity on Mars was also present, Clark said.
He then reminded the audience that terrestrial life often goes dormant for long periods of time in “survival mode.” Clark suggested that life on Mars could be doing the same thing between obliquity cycles. He also said that abiotic photochemistry on Mars could provide reactants to drive chemoautotrophy without photosynthesis. To search for this life, Clark said that you could go vertically down to perhaps subsurface permafrost ice or even a deep liquid groundwater layer. He also said that you could search horizontally, such as looking in salts for salt-tolerant organisms. There are potentially even caves or lava tubes that could support life.
Clark then finished with lessons learned from the Viking missions. Experiments that are simple in the laboratory can be difficult to implement and expensive to fly, and they might have perplexing results. The environmental context is also important. He then said that understanding Mars requires a sample return mission. Lastly, he said that the Viking mantra of “if life is anywhere, life is everywhere” on Mars may be wrong. It is possible that life only exists in certain regions or environments on Mars.
A member of the audience said that it isn’t fair to second guess the Viking experiments, saying that it was one of the great accomplishments of civilization. He did, however, say that a big problem with Viking is that it never detected any of the organics that, at the time, might have been thought to come from meteorites. In a paper he published 20 years ago, the audience member showed that with a rebuilt instrument, if they had been sitting on top of partially oxidized meteoritic organics, they would not have seen them either. He then said that the two camps, the biological and the abiological, are equal, and it really depends on which result they thought was the extraordinary one that needed an extraordinary explanation. He then asked why there hasn’t been a more sophisticated metabolic experiment. Clark agreed with the need for new metabolic experiments on Mars. One hindrance has been planetary protection, but he said that heat sterilization would now only be needed for the sample acquisition hardware in some cases, not the entire spacecraft. He also repeated that it can be done cheaper today.
An audience member then rebutted the claim that Viking did not detect organics, clarifying that it found chloromethanes. Clark said that it was just the cleaning solvent. The audience member responded by saying that if you heat soil with organics in the presence of perchlorates, chloromethanes are seen. They never found them in the blank samples either. Clark said that he was open to that interpretation, but was presenting the Viking results as they were known at the time. The conclusion by the Viking team was that cleaning solvent was used, although its use was never officially documented or confirmed.
Commenting on the frost found by Viking, a participant asked about the role of deliquescence (the process by which a substance absorbs moisture from the atmosphere until it dissolves in it) in terms of potential habitability
on Mars. Clark said that deliquescence was actually predicted. He then said that a deliquescent salt effectively competes against an organism for water. Deliquescence often results in a saturated solution with a water activity below 0.6, which is the most extreme limit known for life. On the other hand, Clark said, maybe deliquescence can attract water even after it becomes saturated, which keeps water nearby that could be used by living organisms. Maybe, he said, life could even outcompete deliquescent salts or they could live in lower water activities than terrestrial life. The same audience member then said that, according to her understanding, the water activity for perchlorate would be too low at <0.6, but for sodium chloride, it would be okay. She noted that this is the habitat found in the Atacama Desert. Clark answered that perchlorates depress the freezing point to about –50°C, whereas sodium chloride only depresses the freezing point to –23°C. At that temperature on Mars, he said, the humidity is still much less than 100 percent. However, a change in obliquity toward warmer conditions would increase the water vapor pressure on Mars, which could make all of these processes easier.
Another audience member mentioned again the likelihood of organic matter from meteoritic impacts, or maybe an igneous, hydrothermal type of abiotic organic matter, which should be all over Mars. Biological organics might also contribute to the organic content on Mars. She said that the Viking experiment would not have broken down all organic macromolecules, because the samples were only heated to 500°C. Therefore, organic macromolecules would not have been detected by Viking. She then said that the release of oxygen complicates the analysis. However, it might be helpful because that means it could combust with the organics allowing for its detection. Clark said that the martian surface should contain organic materials approaching the 1,000 parts per million (ppm) level just from meteorites, judging by both the nickel content of the soil and the impact rates. Therefore, much of it seems to have been oxidized, converted, or degraded to result in the low levels seen today.
A workshop participant asked what the salts (iron sulfates, magnesium sulfates, chlorides, and bromides) found in the Gusev Crater imply about Mars. She said that they have sometimes been interpreted as showing modern mobility of fluids in the top meter of soil. Clark said that the Spirit rover found material that is a mixture of ferric sulfate, magnesium sulfate, and a silica-independent phase at multiple elevations. He has no clue how water could have distributed them this way. Clark also doesn’t know why they haven’t found more concentrated occurrences of chlorine, especially since perchlorates significantly depress the freezing point of water, whereas sulfates do not. This means that the perchlorates and chlorides should be mobile and become highly concentrated, while sulfates should remain more diffuse. However, the opposite is seen. Bromine, he said, is extremely erratic and could be mobilized just by frost, since bromine is also a powerful freezing point depressor.
The last question to Clark was whether he thought that Wolf Vishniac’s “Wolf Trap” should have gone to Mars. The Wolf Trap was designed to place martian dust into a tube containing nutrients in liquid form. Properties such as the pH and turbidity would then be monitored for signs indicating life. However, we now know that martian dust in the atmosphere is in the 3 to 4 micron class, much finer than Earth’s dust. Therefore, suspended dust particles would have confounded the experiment. The question was subsequently addressed when the pH of soil was later measured directly by Phoenix. Vishniac did, however, make an enormous contribution to the thought processes behind the search for life on Mars.
Gary Ruvkun of Massachusetts General Hospital began his talk by showing the tree of life rooted with the universal common ancestor (see Figure 4.2). How this tree was initially constructed, he said, started with trying to isolate common molecules from cells, which began in the pre-DNA era. The ribosome turned out to be one of the most abundant organelles in a cell. Ribosomes are made out of many different proteins and a few different RNAs. The RNAs are either approximately 1,500 or approximately 2,900 nucleotides long. The ribosome is where a piece of RNA made from the genome is decoded three nucleotides at a time to assemble proteins three nucleotides at a time. Ruvkun called the ribosome a living fossil of the RNA world. These molecules were easy to pull out and perform RNA sequencing on, which could be used to infer relationships between different organisms. He likened the process to the way linguists phylogenetically classify languages and the relationships between them to infer how languages evolved from earlier languages.
The History and Current State of Genomic Sequencing
Ruvkun then discussed the modern day genomic landscape. He showed a figure of human ribosomal RNA compared to worm RNA (see Figure 4.3). It showed many regions in the RNA that were identical, justifying the structure of the tree of life (e.g., the animal kingdom on the tree of life is just one small twig). Doing the same analysis between human and archaeal ribosomal RNA, the similarities aren’t as strong, but there are still significant stretches where nothing has changed over the last 3 billion years (Gyr) or so. Ruvkun said that evolution had already perfected this part of the RNA. He then said that ribosomal RNA is the most conserved genetic material in life.
Ruvkun then briefly went through the history of genomics. Ribosomal RNA sequencing began in the early 1970s. The discovery of the Archaean branch of the tree of life occurred then. In 1973, recombinant DNA allowed genes to be created one at a time. Gene sequencing was invented in 1976, allowing for a much faster discovery rate (e.g., approximately 3,000 base pairs of DNA in a typical paper). In the 1990s, this process sped up dramatically with the help of machines, during which time the first organism’s genome, a bacterial genome, was sequenced. In 1997, the first full animal genome was sequenced, a nematode with 108 base pairs. In 2001, the human genome was sequenced (3 × 109 base pairs). Now, Ruvkun’s own laboratory can sequence hundreds of full animal genome sequences (~1011 base pairs) per year at about $100 per genome, compared to about $100 million per genome in 2001 (see Figure 4.4). He said that, although only a small portion of biology has been sequenced, it is a highly diverse portion that allows for the network of relations to be analyzed.
The main machine used for DNA sequencing nowadays is the Ilumina, of which there are about 7,500 in the world. A new genome-sequencing machine by Oxford Nanopore was introduced in 2015. Rather than the 500-pound, power-demanding Illumina machines, the Oxford Nanopore machine has a mass of just 87 grams and can be run on a smartphone. Ruvkun said that there are concerns about its accuracy, but its small size and low power requirement can make it a major asset in genome sequencing. There are already about 1,000 users of it.
There are now about 3,500 eukaryotes with their genomes sequenced, each one having 5 × 106 to 1010 base pairs. Typical animals have 108 to 3 × 109 base pairs. Larger animals’ genomes are usually packed with what is sometimes called “junk DNA,” such as the carcasses of viruses. Out of ~25,000 genes in animals, ~10,000 of them are shared between all the different animal species. There are also more than 12,000 bacterial genomes sequenced
(~4 × 106 base pairs each) and ~700 archaeal genomes sequenced. A typical bacterium has ~4,000 genes. A few hundred genes are universal in all extant life. As an example, he showed a 400 amino acid protein from an animal that encodes a proteasome subunit, a part of an organelle that degrades proteins, and compared it to its counterpart in an archaea. The two were separated for 3 billion years, yet there are still strong similarities. Ruvkun said that it is one of the most conserved proteins in evolution. He then said that there are about 400 genes that are similarly conserved which form the core of biology and were likely present in the last common ancestor of all life on Earth.
Ancient Life and Panspermia
A major problem in the study of evolution, especially early evolution, is determining the time that different branches split off from one another. In the last 500 million years, fossils provide a method to date some branches, but mostly just in the animal kingdom. Fossils from other branches in the phylogenic tree, especially in other domains, are difficult or even impossible to find, particularly as one goes further back in time. However, stromatolites, the fossilized remains of macroscopic mats of bacteria, are known to have existed 3.5 Gyr ago, which is relatively soon after the Late Heavy Bombardment 3.9 Gyr ago, especially for fully developed, rather perfected DNA life-forms. In fact, isotopic evidence for life suggests that life might have existed even earlier (3.9 Gyr ago). By then, life already must have gone through the RNA world, which started out with prebiotic synthesis of proteins and RNA. Life would have already advanced to having cells with RNA as both the coding and catalytic molecule by then. Proteins then took over the catalytic function, and then finally, DNA took over the coding functions. Life, Ruvkun said, already perfected the core biology by about 3.5 Gyr ago. Either it needed to evolve very fast, or it needed to arrive on Earth fully formed already.
A bold belief by Ruvkun is that the tree of life didn’t start ~4 Gyr ago here on Earth, but rather ~10 Gyr ago somewhere else and then later brought to Earth. A weaker statement of panspermia, he said, would be to say that life on Earth may have spread to Mars. Maybe, Ruvkun said, the best way to look for life there is the best way to look for life here. The best way here, he said, is with DNA-based surveys. This is a convenient method because of
the trillion-dollar investment already made in genomics. With the Oxford Nanopore system, Ruvkun said it would be foolish not to use that method first when exploring Mars. To get life from Earth to Mars, meteorite impacts are necessary to eject material from Earth into space. Within about 30,000 years, 0.001 percent of that ejecta can land on Mars.5 Interestingly, that same study showed that 1 percent of the ejecta lands back on Earth, which could potentially repopulate an Earth sterilized by the effects of the meteorite impact.
Ruvkun proposed to take martian soil and extract DNA from it. Similar things are already done routinely on Earth, using soil samples from many different types of environments. One can break open any cells present in a sample of martian soil with an electrical disruptor to release the cells’ DNA. Next, any DNA that is present in this soil is purified on a solid matrix that specifically binds to DNA. There are then standard ways to randomly fragment DNA and put known DNA sequences (linkers) onto the ends of the unknown sequences of the DNA from the soil. One can then transport these DNA molecules, one at a time, through a nanopore—a commercial technology in which the pore’s conductivity is changed as the DNA goes through the pore. By measuring the change in conductivity, each DNA molecule’s nucleotide sequence is obtained. This sequence data is then transmitted to Earth as a megabase file.
Contamination is a major issue, however. He recounted how the Neanderthal genome was sequenced.6 After drilling into the bone and making DNA out of it, 99 percent of the DNA was bacterial. They were then able to
5 B. Gladman, L. Dones, H.F. Levison, and J.A. Burns, 2005, Impact seeding and reseeding in the inner solar system, Astrobiology 5:483.
6 M. Krings, A. Stone, R.W. Schmitz, H. Krainitzki, M. Stoneking, and S. Pääbo, 1997, Neanderthal DNA sequences and the origin of modern humans, Cell 90:19.
pull out the 1 percent that was Neanderthal. Ruvkun said that this example shows that, even if there would be contamination on Mars from Earth life, you would still be able to see a real signal of extraterrestrial genomes. He finished by saying that the Oxford Nanopore technique could even read nonstandard nucleic acids, although its best use would be to search for life as we know it.
A member of the audience commented that he was doing some sequencing for his Ph.D. a couple of years after Viking landed. Working from Ruvkun’s numbers, the Oxford Nanopore system could now do his Ph.D. [research] in 0.086 picoseconds. He then took strong exception to Ruvkun’s claim that the conservation of key genes for billions of years means that it is perfect. The audience member said that it isn’t perfect, but rather, it’s just good enough. An example is the vertebrate retina, which is a bad design (it’s upside down), but it won’t change because it’s good enough. Ruvkun said that he strongly disagreed with that. The audience member then noted that the first genome was sequenced in 1977, the phi X 174 (or ΦX174), a bacteriophage virus.7 Two years later, a paper titled “Is bacteriophage phi X174 DNA a message from an extraterrestrial intelligence” was published, which shows that people have been looking at DNA in terms of astrobiology for a long time.8
Another conference participant reported that, MinION, the Oxford Nanopore’s portable DNA sequencer, has an error rate of 17 percent and asked how it was being improved. Ruvkun said that the 17 percent error rate is per nucleotide. However, there is redundancy built in because you’re running the test many times. The accuracy is only a significant problem if you’re looking for a one base difference, such as a human cancer gene. A high error rate isn’t important if you’re just testing to see whether there is a ribosomal gene on Mars or not. Ruvkun tested it on Bacillus subtilis and was happy with the results. The 17 percent error rate just wasn’t a problem because they got ~100 independent runs of each genomic region.
According to one workshop participant, a ribosomal ancestry reconstruction is characteristically different from protein reconstruction because you never need to put losses into a ribosomal tree. You can, however, always do that with gains, which is almost never the case with a protein reconstruction. This means that there are some things that we don’t know about RNA evolution, both after and certainly before translation. The selectionist interpretation can therefore not always be used for genes, because some of them are locked in at the network level. Ruvkun responded that when he envisions sequencing on Mars, he hopes to find that Mars is stuck in the RNA-world stage. He then repeated that the ribosome is a living fossil of the RNA world; it looks like an RNA replicase would before it could do translation. The transfer RNA adapter molecules were probably replicator molecules that were taking RNA segments. The ribosome, he said, is probably a re-engineered replicase. He said that the RNA world hypothesis is pretty well supported by the discovery of catalytic RNAs. He thinks that the RNA world wasn’t here on Earth like most scientists believe, but rather, it was somewhere else. Ruvkun just thinks that 100 million years is far too short of a time to go from the RNA world to full-on bacteria.
Another member of the audience asked how to use the Oxford Nanopore system in situ on another body’s surface. Ruvkun said it was simple. You just add in a sodium dodecyl sulfate (SDS) solution, a little soap, a hydrophobic disruptor, and then sonicate it. After that, you adhere the DNA and run it into the Oxford Nanopore system. (There is a backup plan if it is RNA.) It’s a robust system, but of course, it would need to be prepared in such a way as to survive months or years in the harsh environment of space.
The Neanderthal DNA (1 percent) from all of the bacterial DNA (99 percent) had implications for planetary protection, one workshop participant said. Ruvkun replied that there is still the issue of bringing organisms to Mars. Ideally, he would like to bring DNA with them as a positive control, potentially synthetic DNA. If the goal is really human exploration, Ruvkun said that they should suspend all planetary protection protocols.
A workshop participant then said that radiation damage on the surface of Mars looks bad for finding DNA there. He then asked how old DNA could be on Mars and still be detectable with the Oxford Nanopore technique,
7 F. Sanger, G.M. Air, B.G. Barrell, N.L. Brown, A.R. Coulson, J.C. Fiddes, C.A. Hutchison, P.M. Slocombie, and M. Smith, 1977, Nucleotide sequence of bacteriophage φX174 DNA, Nature 265:687.
8 H. Yokoo and T. Oshima, 1979, Is bacteriophage phi X174 DNA a message from an extraterrestrial intelligence, Icarus 38:148.
considering that organisms might be more common in Mars’ past than in Mars’ present. Ruvkun said an upper limit on their ability would be finding DNA from about 1 million years ago. However, he wouldn’t bet on the extinction of microbes. He thinks microbes are extremely adaptable and is a proponent of the saying “if life is anywhere, it’s everywhere.” The biggest issue, he said, is whether you’re sensitive to life. With DNA, amplification is easy, but there is still the problem of interpreting it in the presence of a background. They’re not aiming for a fossil though. On the same subject, another audience member said that concentrating a sample to find a cell might be difficult. Ruvkun finished by saying that DNA is the best at concentrating a sample because it can be amplified, although the audience member still questioned the ability to process a large amount of material.
Steven Benner from the Foundation for Applied Molecular Evolution began his talk with the “paradox of molecular signatures” (earlier framed in terms of the “paradox of life” or the “paradox of a biosignature”), which says that a reliable biosignature is a molecular system that cannot arise without life. The paradox is then that this life could never arise. This means that no biosignature can exist to detect life soon after it has arisen—that is, soon after a molecular system has gained access to Darwinian evolution. Instead, we must rely on the subsequent ability of Darwinian evolution to create molecules having structures and complexes that could not possibly have arisen via abiological processes. Darwinian evolution, he said, is a necessary universal feature of life.
The Universal Genetic Biopolymer Structure
Benner then asked what properties a genetic molecule would need for Darwinism to operate on it. He offered one constraint: it needs to be a one-dimensional biopolymer (attempts to assemble a two-dimensional version have so far failed). To support Darwinism, that biopolymer must be able to change its structure to change its information. However, these changes in its structure cannot substantially change its physical and chemical behaviors (e.g., its solubility, molecular recognition rules, and reactivity). For example, in the one genetic biopolymer that we know of, DNA, replacing a guanine with an adenine does not substantially change its physical and chemical properties. Such systems, he said, are rare. Proteins, polysaccharides, and most other classes of polymers exhibit dramatic physical and chemical changes with just small changes in structure. As an example, changing one amino acid in hemoglobin out of 576 causes sickle-cell anemia. In contrast, a biopolymer able to support Darwinism must have fairly constant properties after a change in its information content. Then, under the paradigm of Darwinism, the biopolymer has the ability to be imperfectly replicated, where those imperfections are themselves replicable. This is exemplified for both DNA and RNA, whose properties do not greatly change upon changing nucleotide sequences. Almost all DNA and RNA sequences are soluble in water, bind their complements, precipitate in ethanol, and template polymerases.
The property that makes DNA and RNA special, according to Benner, is that they have a repeating backbone of monopoles. For DNA and RNA, this backbone is the negatively charged phosphate unit. Proteins, on the other hand, have a repeating backbone dipole, which is why they fail as a genetic biopolymer. Evidence for this comes from synthetic biology (also called constructive biology). DNA analogs have been made that do not have a repeating backbone charge. These molecules precipitate and act just like proteins in the sense that their physical behavior dramatically changes if even a single base is altered. A repeating dipole backbone can easily fold, while a repeating monopole backbone prevents folding (see Figure 4.5). A repeating monopole backbone also allows for templating, keeps the DNA soluble, and forces strand-strand interactions to occur at the edges of the nucleobases. DNA’s backbone keeps its bulk molecular properties the same because the monopole backbone is its dominant property. This polyelectrolyte theory of the gene, Benner said, will be true for all life in water throughout the universe. This, Benner said, is not true for biopolymers that lack a repeating backbone charge. Indeed, synthetic biologists have been able to create alternative forms of DNA with entirely different nucleobases, as long as they have retained the repeating backbone charge. In contrast, they have not been able to remove the backbone charges and obtain an evolvable biopolymer.
Conveniently, Benner said, finding polyelectrolytic genetic biopolymers in a sample of water obtained from an alien locale would be trivially easy and, in fact, is the easiest type of potential genetic molecule to find. The
polyelectrolyte will easily bind to a polycharged detector, more easily than other kinds of molecules that carry only a single charge or a small number of charges. As a signaling mechanism, a longer polyelectrolyte will displace shorter polyelectrolytes that are tagged with fluorescence or radio labels. Long polyelectrolytes can be detected in this way even if they are present only at extremely low concentrations in water. After they are detected, they should be examined to determine whether they are random biopolymers or Darwinian biopolymers. Key features to indicate Darwinian biopolymers are homochirality and being built from a “controlled vocabulary” (a small set of building blocks). Thus, this model for the universal life detection system assumes only the universality of Darwinism and the polyelectrolyte theory of the gene based on one-dimensional biopolymers.
The rest of the genetic biopolymer, Benner said, could be anything. However, ribose is clearly one of the best backbone molecules that have been examined, particularly in terms of molecular recognition, although a handful of other possible structures have been found to work analogously (threose and some bicyclic structures are especially worthy of note). For this reason, most of the prebiotic chemistry community focuses on ribose RNA. While there is not a clear path to get RNA as the first Darwinian biopolymer, Benner said that a path is likely to be found soon, since work over the past decade has offered solutions to many of the problems that were considered insurmountable in the RNA-first model for the origin of Darwinism. One problem arises from the general observation that applying energy to organic matter of the sort that cannot undergo Darwinian evolution just produces tar (i.e., heating up sucrose makes caramel). Additionally, it is hard to obtain an available form of phosphate, especially in the presence of calcium. Further, RNA is unstable in water. Water is essential for life, but it also destroys the RNA biopolymers.
He then said that the C=O group in an organic molecule is a source of horrible reaction complexity that leads to such tars. Ribose has a C=O group, enolizes, reacts with itself, and then forms tar under alkaline conditions. Experiments show that ribose forms tar at pH 7 at 50°C after just 7 years, which was said to preclude the use of ribose and other sugars from being components of the first genetic material.9 However, mineralogy can mitigate this problem. Borate, Benner said, is a poor mineral-forming element, is concentrated in residual melts and igneous rocks, and is easily leached from these rocks by erosion into aquifers. Sugars have adjacent hydroxyl groups, which borate binds to in an extremely stable way. Borate thus binds to ribose, removing the C=O group and preventing ribose from becoming tar, thus allowing it to accumulate. Further, borate also guides the chemical reactions of smaller carbohydrates that deliver 5-carbon species, including ribose.
Benner then described recent literature that supports a discontinuous model for RNA synthesis. The model starts with gases (CO2, H2O, N2, and CH4) that can be converted by UV light and electric discharge to give hydrogen
9 R. Larralde, M.P. Robertson, and S.L. Miller, 1995, Rates of decomposition of ribose and other sugars: Implications for chemical evolution, Proceedings of the National Academy of Sciences of the U.S.A. 92:8158.
cyanide (HCN), cynamide (HNCNH), and formaldehyde (CH2O), all generally agreed to have been available on the early Earth. Borate moderates the early chemical process that converts formaldehyde to ribose borate. Cyanide and cynamide are hydrolyzed to formamide and urea.
However, all this requires dry land. The entire process is defeated by dilution in a global oceanic system. That means that a submarine origin of life, as in hydrothermal vents, must solve the problem about dilution and the instability in water of many of the bonds in RNA. Benner said that a desert with occasional intermittent water might be ideal. He then referred back to a previous talk about Mars’ changing obliquity, which would produce exactly the type of intermittency they need.
The two conditions Benner requires for life, borate and intermittently wet deserts, may not have been available on early Earth, according to some geologists. Further, several geologists have argued that, absent a history of plate tectonics, boron could not have been concentrated in the lithosphere sufficient to attain productive concentrations anywhere on early Earth. Efforts to find residual soluble boron minerals in ancient rocks are not likely to be directly successful, because kernite, ulexite, colemanite, and other boron minerals are not expected to survive for a long time. These and other borate minerals are unstable to metamorphosis, yielding monazite, apatite, and tourmaline. However, we can look for these derived borate and phosphate minerals. For example, a 3.8 Gyr old rock was just recently found to contain monazite, apatite, and tourmaline—suggesting the existence of borate prior to their metamorphic alterations.10,11
Benner then addressed the possibility that the inventory of water on early Earth required an ocean world with no dry land. Without dry land, there could be no desert, no borate evaporites, no ribose, and no RNA. If this was so on Earth, the borate-involving prebiotic chemistry should nevertheless have been possible on Mars, which almost certainly had less water. Benner said that there could even be borate-ribose on Mars today. John Grotzinger’s earlier talk (see Chapter 2) showed that everything Benner thinks needs to be there for life to arise was actually on Mars. This includes opal CT; Elisa Biondi in Benner’s group recently found evidence that RNA adsorbs onto opal in stable form.
Moving to the problem of phosphate concentration, Benner referenced records showing contemporary precipitation of gypsum (CaSO4•2H2O) and lüneburgite (Mg3B2(PO4)2(OH)6•8H2O) on Earth. This observation is significant with respect to the availability of phosphate and complex geological environments. In simpler environments, when Ca+2, Mg+2, PO4-3, and SO4-2 are interacting together in the absence of borate (BO3-3), they form apatite (composed of Ca+2 and PO4-3) and epsomite (composed of Mg+2 and SO4-2). Apatite sequesters phosphate, but largely unproductively. In the presence of borate, however, they form gypsum and lüneburgite. This keeps phosphate from being locked away via calcium capture. The phosphate instead joins with the borate into borophosphate. When ribose encounters lüneburgite, it extracts the boron, which disrupts the mineral and releases phosphate. The phosphate is then available to phosphorylate the nucleoside. With the borate coordinated to a specific pair of OH groups, the only products are the five prime phosphorylate ribonucleosides. Without the borate present, many ribonucleoside phosphorylation products are seen in laboratory settings. That all this corresponds with martian geochemistry makes Benner think that panspermia from Mars to Earth is at least plausible, especially if the absence of desert land on early Earth requires this prebiotic chemistry be on Mars. It should be noted, however, that recent work of Stephen Mojzsis supported by the Foundation for Applied Molecular Evolution-Templeton program found that the amount of dry land on early Earth may be sufficient so as to not enforce this requirement of panspermia.
Moving to a different type of chemistry, Benner said that the main problem with life forming in non-aqueous environments, like Titan’s hydrocarbon lakes, is solubility. A biopolymer with a backbone of repeated charges will not dissolve in hydrophobic solvents. A potential solution is a repeating dipole that presents only the same end of the dipole all along the biopolymer (e.g., polyethylene glycol). A molecule like this might be able to prevent aggregation and folding while maintaining a genetic function. However, cryosolvents are bad solvents because
10 E.S. Grew, R.F. Dymek, J.C.M. De Hoog, S.L. Harley, J. Boak, R.M. Hazen, and M.G. Yates, 2015, Boron isotopes in tourmaline from the ca. 3.7-3.8 Ga Isua supracrustal belt, Greenland: Sources for boron in Eoarchean continental crust and seawater, Geochimica et Cosmochimica Acta 163:156.
11 S. Mishima, Y. Ohtomo, and T. Kakegawa, 2016, Occurrence of tourmaline in metasedimentary rocks of the Isua Supracrustal Belt, Greenland: Implications for ribose stabilization in Hadean marine sediments, Origins of Life and Evolution of Biospheres 46:247.
they’re cold and therefore relatively insoluble. They are unlikely to work on Titan, but they might work on a warm Titan with oceans of hexane or octane.
Disequilibrium and the Limitations of Darwinism
Benner’s last point related to a claim often made in astrobiology that disequilibrium can be used as a biosignature. For example, a forest in an atmosphere of oxygen is a disequilibrium that can be interpreted as a biosignature. However, Benner said, the disequilibrium exists because Darwinism has been ineffective at creating enzymes that catalyze the destruction of cellulose. Indeed, this ineffectiveness is illustrated by the abundance of uneaten beds of coal. More than a dozen different cellulase families appear in a range of organisms. However, they all appear to have evolved from previously created enzymes that initially had other roles. Darwinism has not created a macroscopic life-form able to exploit the energy in the forest effectively, which illustrates a possible limitation of Darwinism.
One solution to this problem, Benner said, is Lamarckism. For example, humans learned how to use cellulose about 2 million years ago by learning how to build fires and burn wood, and then transmitting this skill to their children not by DNA, but rather by teaching. Benner said that any alien life smart enough to talk to us, or especially to travel to Earth, would also have discovered Lamarckism. Indeed, it would have gained Lamarckian control over its molecular biology, eliminating Darwinism as a mechanism for preserving its core genetic capabilities or creating new ones. Clustered regularly interspaced short palindromic repeats (CRISPR) and germline gene therapy puts humans on the verge of this evolutionary change. Therefore, Lamarckism will have its own biosignatures. For example, carbon-fluorine bonds cannot be generated either biologically or abiologically, but can be manufactured by an intelligent life form. In other words, intelligent life will eschew Darwinism and instead use Lamarckism. This means that the search for extraterrestrial weird life does not necessarily require understanding its unknown molecular biology.
Benner then presented his conclusions. On a planet with water, any life will have a genetic biopolymer with a backbone of repeating charges. This type of molecule can be easily concentrated from alien aqueous environments, such as from the plumes of Enceladus, in order to be detected with today’s technology. Further, once concentrated, it can be easily detected in situ. Finally, downstream analysis of its structure will allow us to directly determine whether it is the product of Darwinism or a random, accidental polymer.
There is not yet a reason to believe that RNA is the universal structure, but on a planet with similar geology to the Earth or Mars, it might be likely. However, Benner thinks that, with a desert and an appropriate mineralogy, the abiotic synthesis of RNA will not be an unsolved problem for much longer. He also said that a good non-aqueous environment for life has not yet been discovered and that cryosolvents are particularly bad. Benner said that disequilibrium implicates the impotence, or even the absence, of Darwinism. It is not, if examined out of context, a biosignature. Lastly, Benner said that intelligent life that can move beyond Darwinism to Lamarckism would have its own set of biosignatures, regardless of the underlying molecular biology.
A member of the audience said that she liked Benner’s comparison between the calcium phosphate and the magnesium borate. The geochemical principle behind this, she said, is called the “geochemical divide.” She then said that another anion at the time would have been carbonate. She would be interested in an experiment that added various quantities of carbonate to the system to see how that would affect the types and ratios of different minerals precipitating from it. Benner started by saying that we know very little about the early Earth’s geology. However, he thinks that it was the borate that was scarce in the early Earth. He then said that people have studied systems with multiple ionic inputs and that he was open to collaboration on future studies.
Emphasizing a caveat to Benner’s favorable view of martian geology in terms of the origin of life, another audience member said that the data is not necessarily representative of the entire planet. Gale Crater, for example, had multiple episodes with water and minerals precipitating through it. There are therefore questions about how this stuff formed and whether it was in association with other materials there. She thinks that maybe new types
of work on Mars might be necessary to see whether all these necessary minerals were present and available at the same time sometime in Mars’ past. Benner said that he is aware of the environmental heterogeneity on Mars. However, he’s ready now to simulate Mars with various minerals to see how hydrogen cyanide and formaldehyde reacts in it. Benner said that doing these experiments on manufactured minerals is required because even samples from Death Valley would contain enough life to eat all the organics they gave it. He said that he’s tested their ability to synthesize minerals using 50 rocks from the “Benner Collection of Fine Rock Specimens” in order to show that his synthesized rocks are comparable to natural rocks.
A workshop participant then said that the Mars teams have found mobile phosphate (in the sense that its concentration changes over short distances) in multiple cases. The Curiosity team has also discovered boron in higher than expected concentrations. He then finished saying that all the components to develop life are there.