Life Detection with the Enceladus Orbiting Sequencer Christopher E

Home , Graphene

Life Detection with the Enceladus Orbiting Sequencer Christopher E. Carr & Maria T. Zuber Gary Ruvkun Massachusetts Institute of Technology, Department of Massachusetts General Hospital, Dept. of Mol. Biology Earth, Atmospheric and Planetary Sciences Harvard Medical School, Dept. of Genetics Cambridge, MA 02139 Boston, MA 02114 [email protected], [email protected] [email protected]

Abstract— Widespread organic synthesis in the early solar RNA or DNA on Mars due to the significant meteoritic nebula led to delivery of similar complex organics, probably transfer between Earth and Mars [1, 9-11]. However, the including nucleobases or their precursors, to many potentially ability to detect and sequence other nucleic acids and more habitable locations such as Mars, Europa, and Enceladus. If generic IPs would provide additional sensitivity. We review life evolved beyond Earth, the presence of these organics could prospects for extant life on Enceladus and describe an have biased life towards utilization of informational polymers (IPs) like RNA or DNA. Given this, searching for and instrument concept specifically geared to searching for life sequencing any such IPs offers a definitive, information rich, on this remarkable icy moon. approach to life detection that complements existing methods. Saturn’s icy moon Enceladus offers possibly the best conditions in the solar system to find extant life beyond Earth. 2. IS ENCELADUS HABITABLE? Recent discovery of a salt-water plume likely derived from sub-surface liquid reservoirs provides direct access to this A Brief History of Enceladus potentially habitable environment. We describe an instrument Enceladus is a volatile-rich (density 1.6 g/cm3) moon concept, the Enceladus Orbiting Sequencer (EOS), specifically geared to search for life on Enceladus. As a payload on board located in Saturn’s E-ring (Fig. 1A). Enceladus likely an Enceladus flyby or orbiter mission, EOS would capture ice formed from icy planetesimals produced in the solar nebula grains from the plume, then concentrate and characterize any that were then partially devolatilized during their long charged polymers using nanopore or semiconductor consolidation in the Saturn subnebula [12]. Its icy shell may sequencing. Searching for life on Enceladus could give us our cover a subsurface ocean, which may be in direct contact first glimpse of a second genesis and test whether biochemistry with its rocky core (Fig. 1B). Plume ice from Enceladus is varied or universal. feeds the Saturn E-ring, which has been observed to be stable since the 1960s. As Enceladus sweeps counter- TABLE OF CONTENTS clockwise around Saturn and through the ring, it collects irradiated ring particles and leaves a shadow (Fig. 1C). 1. INTRODUCTION ...... 1 2. IS ENCELADUS HABITABLE? ...... 1 3. MISSION ARCHITECTURE ...... 4 4. EOS INSTRUMENT CONCEPT ...... 5 5. ICE GRAIN CAPTURE ...... 6 6. INFORMATIONAL POLYMER SEQUENCING ...... 6 7. SUMMARY AND CONCLUSIONS ...... 8 ACKNOWLEDGEMENTS ...... 8 REFERENCES ...... 8 BIOGRAPHY ...... 10

1. INTRODUCTION Recent findings of nucleobases or their precursors on meteorites [1, 5] and in interstellar space suggest that life Fig. 1. Saturn's icy moon Enceladus. (A) Ice from beyond Earth could be based on nucleic acid-like polymers Enceladus forms Saturn's outermost ring. (B) Enceladus has such as RNA or DNA. Furthermore, complex organic a rocky core of radius 150-160 km, covered by 90-100 km synthesis is widespread in stellar nebulae [3, 6], likely of H2O. (C) Enceladus both creates and sweeps through the around all stars. As a result, common sources of organic E-ring. The bright dot is the plume, whereas the moon is a material were delivered to multiple potentially habitable tiny black region above the bright dot. (D) The surface of zones in our solar system, such as Mars, Europa, and Enceladus has geologically young, fractured (blue) regions Enceladus. This delivery could have biased the development and older, cratered regions. (E) Cassini identified the of life towards similar biochemical solutions such as Enceladus plume during a 2005 flyby. All images from utilization of informational polymers (IPs) like RNA and NASA Cassini mission (CICLOPS); panel A is contrast DNA. An even stronger case can be made to search for enhanced, B is artist's impression, D and E are false color.

The pattern of surface modifications, from the heavily- cratered north to the fractured almost un-cratered south polar terrain (SPT, Fig. 1D, lower right), implies episodic geologic activity over a 4 billion year period [13]. Also indicative of geological activity is the moon’s limited topographic relief [14], which is unusual for small, low gravity (0.1 m/s2) satellites. While originally considered too small to be active, Cassini flybys identified the geologically young (<1 million years) SPT, associated with elevated temperatures, tectonic rifts, and ice jets [13] (Fig. 1E).

Plume Characterization Fig. 2. Plume sources and likely structure. (A) The four Imaging of the SPT revealed four main subparallel furrows major sulci are associated with eight plume sources (yellow and ridges (sulci), each about 130 km in length with central points) and large heat anomalies (overlay). (B) Damascus two km wide depressions. These sulci are associated with Sulcus, relief 10x exaggerated. (C) Plume source model: I, heat anomalies and eight known plume sources (Fig. 2A-B). II, III indicate predominant ice grain type. Image credits NASA/JPL/GSFC/SwRI/SSI for A and B. Ground-based spectroscopic analysis of the plume, including gaseous components, argued for low sodium levels consistent with a deep ocean, a freshwater reservoir, or ice [15]. However, the Cassini Cosmic Dust Analyzer related to life on Earth, unless life on Earth and Enceladus (CDA) revealed a population of sodium-rich (0.5-2%) both derived from another (outer planets or beyond) source grains in the plume, consistent with a subsurface ocean [16] that had significant meteoritic transfer to both bodies. and an explanation for the lack of salt in the plume vapor: sodium salts would remain in the liquid phase as the water Liquid water?—Despite the generally frigid surface freezes out as part of the ice crust, with sublimation of this temperature (-200˚C) of Enceladus, several plume and crust yielding plume gas. Later flybys and analysis by the surface characteristics argue for a liquid water source for at CDA strengthened the case for a subsurface salt-water least part of the plume. First, the substantial heat output of reservoir [17], consistent with models of a subsurface ocean the SPT, at 16 GW [19], is most consistent with an elevated in contact with the moon’s rocky core (Fig. 2C). temperature source. Second, modeling of plume ice particle growth suggests source temperatures of at least 190K [18]. To date, observations and models argue for three general Third, the presence of sodium ice grains requires rapid types of ice grains in the plume [17]: Type I grains are freezing, consistent with quickly cooled liquid water. small, salt poor, and likely formed from nucleation of gas Fourth, the fracture patterns in the south polar terrain are phase water vapor, leading to high ejection speeds and best explained by a global liquid ocean [20]. Of note, explaining their dominant contribution to the E-ring. Type II methanogens in permafrost can stay metabolically active at - grains contain organic and/or siliceous material, might 20˚C by utilizing tightly bound unfrozen water [21]. indicate surface/ocean communication, and, speculatively, could be derived from solid clathrates [18]. More organics Essential nutrients?—Enceladus acquired organic material are observed in the center of the plume [17]. Type III grains as a consequence of its primordial origin, and by delivery of are salt-rich larger particles found in higher densities in the organics by comets and meteors. Some organic material has lower part of the plume, and presumably formed from a been definitively detected in the plume [17]. The organics rapidly frozen spray of salt water. They constitute 70% of available to any origin of life on Enceladus may have even the grains above 0.2 μm in size, have lower ejection speeds, included the building blocks of life as we know it (see Why and dominate the mass flux (99%). search for nucleic acids and related polymers? below).

Extant Life? Energy source? — Any life at Enceladus is unlikely to use photosynthesis due to low light levels and the inability of If extant life exists on Enceladus, it either evolved there (a light to penetrate into the subsurface. Similarly, oxidative second genesis) or was carried there from somewhere else phosphorylation appears unlikely. The most likely model for (panspermia). Either way, life as we know it requires liquid any life on Enceladus is chemolithotrophy. water, essential nutrients (mainly made of the CHNOPS elements), and a source of energy (chemical or redox Ecosystems that apply to Enceladus (depending neither on gradient). If we are to posit a second genesis on Enceladus, photosynthesis nor on oxygen) include methanogens that it must also have had time to evolve. We address these consume H2 produced by serpentinization (iron oxidation by considerations in turn. water) of olivine in the Columbia River basalts [22], and sulfur-reducing bacteria that utilize H2 generated as a Panspermia?—Extremely limited meteoritic transfer byproduct of radioactive decay [23, 24] generating H2S. between the inner planets (including Earth) and Enceladus Deuterium-to-hydrogen (D/H) ratio measurements may help virtually guarantees that any life on Enceladus will not be determine if the source of methane in the plume is derived 2 from serpentinization or is primordial in nature [12]. McKay we may be able to apply them to detection of life on et al. [21] also describe a hypothetical methane cycle on Enceladus. Enceladus in which methanogens consume H2 and the CH4 is thermally processed in hot rock to H2. They also suggest Why search for nucleic acids and related polymers? that a very low ratio (0.001) of non-methane hydrocarbons All known life utilizes deoxyribonucleic acid (DNA, Fig. to CH4 may indicate a biological CH4 source. 3A) or ribonucleic acid (RNA, Fig. 3B), a linear polymer made from nucleotides, each one composed of a backbone Some have argued that the biggest redox limitation for molecule (phosphate group), a nucleobase, and a tripartite Enceladus is a lack of oxidants [21]. However, at least two group (sugar) linking the base and adjacent backbone potential redox cycles are available: turnover with the molecules. The pattern of nucleobases directly encodes the surface, bringing a supply of oxidants to a sub-surface enzymatic activity of RNAs and indirectly encodes the ocean, and thermal reformation of chemicals at depth amino acid sequence of proteins through the genetic code, (described above). which maps triplet codons to a smaller group of amino While Europa’s surface ice is heavily irradiated, radiation acids. levels at Enceladus are much lower, limiting the available Nucleotide structure is likely a consequence of common oxidants at the surface. However, the E-ring may act as a building blocks: Presence of the ribose precursor chemical processor [25], providing an extended surface to glycolaldehyde in interstellar space [30] could have led to generate oxidants that are later collected by Enceladus. the development of IPs based on glycol (GNA), threose Turnover at active sites could bring surface oxidants into (TNA), or ribose (RNA/DNA) sugars (Fig. 3C). Charged contact with a sub-surface ocean and help maintain redox backbones are likely universal for aqueous life [31]: they gradients. separate the physical properties of the polymer from its Enough time to evolve life?—Most current models of the associated information content, facilitating replication and moon’s energy budget cannot explain the large heat evolution. This places limits on possible backbones other anomaly associated with the plume. For example, radiogenic than phosphate. In addition, meteorites delivered phosphide heating can explain less than 1 GW. One possibility that minerals to habitable environments, including Earth [32-34], does explain the heat anomaly is that of global tides from a where they may have supplemented a meager terrestrial shallow (<10 km) ocean [26]. Another possibility is that the supply of phosphorus. But what about the third nucleotide current heat output of Enceladus may be above the steady component: Might life beyond Earth use non-standard state value. How can this be explained? nucleobases? Current evidence suggests perhaps not.

Enceladus may undergo periodic oscillations in surface heat Widespread synthesis of complex organics occurred early in flow due to changes in orbital eccentricity, just as does Io the history of the solar system in the solar nebula [6]. Lab [27]. Io is predicted to have peak surface heatflow that lags experiments (Fig. 3D) reveal the formation of amino acids changes in eccentricity. While the current heatflow on and nucleobases [3, 4] including adenine (A), cytosine (C), Enceladus seems unsustainable in comparison to its current guanine (G), and thymine (T) (the standard bases in DNA, orbital eccentricity (0.005), this could be explained by past which form A-T and C-G base pairs through hydrogen higher eccentricity [21]. This raises important questions: If bonding) and uracil (U) (which substitutes for T in RNA). activity on Enceladus is periodic, how long does it last? One Thus, widely mixed in the nebula, the building blocks of life hint is that it may take 30 million years to freeze out an as we know it were likely delivered to all potentially Enceladus subsurface ocean [21, 28]. If Enceladus is habitable zones in the solar system via comet and meteor habitable during periods of high heat flow, could life impacts. Analysis of meteorites (Fig. 3D, blue bars) has survive during periods of reduced activity? Or could it confirmed the presence of extraterrestrial amino acids and evolve during one period of activity? nucleobases [1, 5, 35-37] including G, hypoxanthine, xanthine, A, purine, and diaminopurine [1]. Searching for life on Earth in isolated subglacial lakes may inform our search for life on Enceladus. Preliminary results Non-standard bases play important roles in life today. For from the Russian drilling expedition that has drilled through example, hypoxanthine is part of the inosine (I) nucleoside 3.5 km of ice into Lake Vostok, a sub-glacial Antarctic lake (a nucleobase linked to a sugar backbone) found in transfer thought to be isolated for millions of years, has not yet RNAs, where it is essential for proper translation of the found cells above the background level in their clean room genetic code (from 64 base triplet codons to 21 amino acids) (10 microbes/ml); most of the cells have been identified as in wobble base pairs through promiscuous pairing with A, contaminants from drilling oil (Nature, Brian Owens, 18 Oct C, or U [38]. Thus, the error-correcting capabilities of the 2012). Microbes have been identified in other isolated genetic code may be enabled by extraterrestrial chemistry. subglacial lakes [29], where they are thought to have Another example is xanthine, which is found in many migrated from the subsurface. human tissues and plays a role in generation of free radicals. Just as we can apply exquisitely sensitive molecular biology In addition, diaminopurine base pairs with both xanthine techniques to the detection of life in these environments, so and T (substituting for A and leading to increased basepair 3

Fig. 3. Why search for nucleic acids and related polymers? (A) DNA double helix. (B) Components of a nucleotide. (C) Potential evolution of nucleic acid polymers from interstellar glycolaldehyde; (S)-GNA (but not the (R)- stereoisomer) binds RNA, while TNA binds RNA or DNA. (D). All standard and some non-standard nucleobases have been identified in meteorites [1, 2] (blue) or lab simulations of the solar nebula [3, 4] or of a reducing planetary atmosphere (Titan [8], red).

stability; cyanophage S-2L uses this property to facilitate from the surface most likely using a drill. Similarly, host evasion [39]). Thus, these nonstandard base pairs are accessing habitable zones on Europa may require drilling not foreign to biology, but are in fact used by it today. through an unknown layer of ice to reach the subsurface ocean. Thus, life detection missions on Mars and Europa are Further synthesis took place in reducing planetary not only technically complicated but impose a higher risk of atmospheres (early Earth, Mars, Venus and present-day forward contamination of potentially habitable zones. Titan). For example, Cassini measurements have identified Enceladus flyby missions offer a variety of end-of-life unknown large organic molecules in Titan’s upper options, while orbiter missions could deorbit onto Enceladus atmosphere; lab-based experiments (Fig. 3D, red bars) (with obvious planetary protection issues) or leave orbit yielded 18 molecules corresponding to amino acids and all using minimal delta-v and exercise other options. of the standard nucleobases [8]. Thus, the basic building blocks of life as we know it may form in reducing planetary Key trades: Flyby or Orbiter? In-situ vs. Return? atmospheres, in interstellar space, or, as has long been A key trade that must be pursued is to determine whether known, through aqueous chemistry. Finally, the availability volatiles, and in particular, intact nucleic acids or related of common building blocks in multiple habitable zones may informational polymers, can be recovered from plume have biased “independent” genesis events towards the use of particles at sample capture velocities consistent with flyby similar informational polymers (IPs). missions. If not, an orbiter mission would be required, with the commensurate cost of a propulsion system with the required delta-v to achieve Enceladus orbit. 3. MISSION ARCHITECTURE Unlike Mars and Europa, brine from the interior of Another key trade is whether to carry out extensive in-situ Enceladus can be acquired while orbiting or possibly during analysis or carry out a sample return mission. The baseline a flyby, due to the geysers erupting from its surface. Like cost of a sample return mission may make it prohibitively Mars, but unlike Europa, Enceladus also presents a more expensive. In addition, a sample return mission to moderate radiation environment without extensive ionized Enceladus would likely be classified as planetary protection trapped particles, a benefit to radiation-sensitive category V (restricted), due to the potential for chemical instrumentation. Enceladus also enables us to access the evolution and/or life on Enceladus. Under this planetary interior without a high risk of forward categorization, the cost associated with planetary protection contamination [18]. This is unlike Mars, where potentially could potentially exceed the baseline sample return mission habitable environments would need to be directly accessed cost [40]. In addition, during the return trip of up to 5 years 4 in one proposal [40], samples would be exposed to space decrease aerogel density down to 2 mg/cm3 to facilitate less radiation and would otherwise need to be preserved under destructive sample capture, and collect and secure volatiles. pristine conditions. We propose to re-seal compartments to protect against Thus, even aside from the planetary protection-associated volatile loss and possibly allow multiple sampling events for costs, we argue for at least some in-situ analysis, to facilitate a single compartment. We assume that the shock pressures use of fresh samples and to reduce the risks of involved will be enough to disrupt any cells in plume contaminating the samples with Earth organics. particles to enable later release of genetic material (informational polymers, IPs). This assumption should be Plume Sampling tested experimentally. Additional lysis steps can be taken at Several post-Cassini follow-up missions to study Enceladus the cost of greater complexity. have been proposed. Two (Titan and Enceladus $1B Once ice grains have been collected, any IPs must be greatly Mission Feasibility Study and the Enceladus Flagship concentrated to cope with extremely low sample volume Mission Concept Study, reviewed by Tsou et al. [40]) have and facilitate low downstream volumes. The top candidate extremely high sample capture speeds of 7 to 10 km/s, even for this is Synchronous Coefficient of Drag Alteration higher than the Stardust mission (6.1 km/s). The slowest (SCODA) [41, 42], which can achieve concentration up to Cassini relative velocity to date was 6.2 km/s (flyby E-13 on 5000X, while also demonstrating extreme contaminant December 21, 2010, out of 19 total flybys through October rejection (Fig. 4B). On a related note, the Enceladus Amino 2012). Recently, Tsou et al. [40] proposed a trajectory that Acid Sampler, a NASA ASTID-funded project, will attempt could achieve Enceladus flybys at reduced speeds, as low as to demonstrate a 104 concentration factor. 3 to 4.5 km/s. Plume velocities could modify the actual sample capture velocities, but many of the particles can be Concentration of nucleic acids via SCODA relies upon the sampled in a stagnation region before they fall back to the physical properties of long polymer chains, and specifically surface of Enceladus. Therefore the plume velocity can be upon their inability to quickly reorient after directional ignored to first order. changes in the electric field. Four electrodes are used to apply rotating dipole and quadruple electric fields to Once in orbit, sampling could be accomplished at much generate a divergent velocity field, allowing for selective lower speeds and higher sampling rates. For example, at an concentration of long charged polymers at the geometric 80 km orbit, orbital velocity is only 147 m/s, and the center of the electrodes when the time-averaged electric sampling frequency could be as high as 6 times per day field is zero. While long charged polymers focus, other instead of Cassini’s average of a few times a year. charged or neutral particles do not, yielding selective For either an orbiter or flyby mission, orientation control focusing of polymers like, but not limited to, RNA or DNA. would be important to align the desired ram direction with For DNA, polymers longer than 200 to 300 bp are focused, the particle flux, which is approximately 1 ice particle per equivalent to a few times the persistence length. SCODA cubic meter at ~80 km orbit [40]. Lower orbits may be has been commercialized by Boreal Genomics and has required to acquire adequate materials for in-situ analysis demonstrated high efficiency in low abundance samples. while avoiding loss of volatiles during repeated exposure of a sampling system.

4. EOS INSTRUMENT CONCEPT Ice grain capture is a central challenge of any Enceladus flyby or orbiter mission that seeks to preserve organics for in-situ analysis or sample return. Addressing this challenge may enable a variety of potential downstream applications.

Similar to other missions, the basic concept is to use aerogel to decelerate ice grains and capture them (Fig. 4A). One or more compartments in an array would be opened during a specific flyby or sample collection orbit. Given known approximate sample capture velocities, one can design aerogel densities and thicknesses to “encourage” final deposition of particles in an agarose layer. In fact, agarose- Fig. 4. EOS sample acquisition. (A) Ice grains decelerate based aerogel (SEAgel, US patents 5,382,285 and through aerogel and deposit into agarose. (B) SCODA [41, 5,360,828) can be made with densities as low as 1.5 mg/cm3 42] permits focusing of generic charged polymers to achieve (versus 1.25 mg/cm3 for air). Tsou et al. [40], in their massive sample concentration and contaminant rejection. proposed LIFE Enceladus sample return mission, would Image credit: Boreal Genomics.

Once the informational polymers have been focused, they that seek to avoid the assumption that life elsewhere may can be characterized through sequencing. We now discuss share any particular features with life on Earth are not as issues relating to ice grain capture and then discuss sensitive, nor do they provide definitive answers. Two approaches to sequencing of informational polymers. examples include mass spectrometry or microcapillary electrophoresis with laser induced fluorescence [47].

5. ICE GRAIN CAPTURE Other approaches? The ice grains emitted by the geysers (up to 100 μm Mass spectrometry (MS) can be used for sequencing of diameter) are large enough to harbor microbial cells (2 μm proteins or nucleic acids, but is generally limited to lengths typical) and the largest grains are negatively charged and of less than 30 base pairs (bp) [48]. In addition, MS is not carry 90% of electrons in the plume. Collecting these particularly sensitive and is thus limited to identifying impacting grains while leaving any IPs adequately intact molecules of relative abundance. MS was studied may pose the biggest challenge. intensively for DNA sequencing during the human genome project and rejected in favor of more successful alternatives. The temperature rise as a function of velocity clearly favors MS has been more successful in protein sequencing, due to an Enceladus orbiter. For example, at the Stardust collection the tendency of nucleic acid polymers to fragment or speed (6.1 m/s), the temperature rise for an ice grain would undergo base loss during ionization. Thus, mass come to nearly 2 x 104 K (assuming all kinetic energy is spectrometry is not currently a viable option for in situ spread evenly over the ice grain and using the specific heat sequencing of non-trivial length nucleic acid polymers, of water). Reducing the velocity to 200 m/s gives a more particularly when abundances are low. moderate 20 K. In practice, much higher velocities may be possible due to uneven heating of grains: gradients as large Microcapillary electrophoresis with laser induced as 2500 K/μm have been estimated for experimental studies fluorescence (μCE-LIF) is a sensitive (parts per trillion, ppt) of hypervelocity capture of meteorite powders by aerogel way to identify amino acids, nucleobases, and other [43]. Ice grains are unlikely to support gradients this large molecules, and is being developed for in situ use [47]. The but may still undergo extremely uneven heating. target molecules are extracted and labeled with a fluorescent probe, then electrophoresis is used to separate the targets by Experimental testing of ice grain capture is clearly required. size. μCF-LIF could be used with Sanger sequencing to Some Stardust aerogel tracks end without dust particles, separate labeled DNA fragments, generating one sequence indicating potential loss of volatile material. Recently, a per separation. While sensitive, μCE-LIF is less sensitive two-stage light-gas gun [44] has been used to study whether than sequencing and cannot efficiently yield large numbers ice grains could be captured by aerogel at stardust velocities of reads. In contrast, sequencing can generate a nearly of 6.1 km/s [45]. Analysis of impact tracks suggested no complete genome from a single cell [49]. If a cell has a terminal grains, consistent with loss of volatile material. single-copy 5 Mb genome (5 femtograms DNA) and is Tests at lower velocities such as 2 to 4.5 km/s should be captured into 1 μl of water, detection corresponds to 5 parts carried out to ascertain whether capture of relatively intact per trillion sensitivity. The actual sensitivity is higher, ice grains is feasible for an Enceladus flyby mission. because sequencing of this cell will involve ~1 M independent sequencing reads. For an orbiter mission, it may be possible to use electrostatic focusing of large negatively charged grains to Sequencing nucleic acids and related polymers dramatically increase the collection efficiency and focusing Until recently, sequencing instruments have been large, of grains. Of note is recent progress in this area for airborne heavy, complex, and required specialized reagents and particle collection [46]. optics. However, it is now possible to consider in-situ massively parallel metagenomic sequencing on a

commercially produced Ion Torrent solid-state chip [50]. 6. INFORMATIONAL POLYMER SEQUENCING This complementary metal-oxide semiconductor (CMOS) Why sequencing? chip is similar to those found in digital cameras, but instead of capturing light, it detects pH changes resulting from If life beyond Earth uses nucleic acids, either because it is addition of bases to growing DNA chains. Current related or because such life evolved from common generation chips have from 1.2 M to 165 M wells. This precursors to use similar informational molecules, we can enables concurrent sequencing in millions of wells in leverage the powerful tools that have emerged from billions parallel, requires no imaging or optics, and is extremely of dollars invested in genomics that are now found in small, fast, and robust. thousands of labs and will soon be ubiquitous in health care and beyond. To use this semiconductor sequencing chip (Fig. 5A), adaptors are ligated to end-repaired fragmented DNA to Sequencing is sensitive down to a single molecule and is make a sequencing library, which is amplified through highly specific: there are no known abiotic routes to nucleic emulsion PCR and enriched to give positive beads, each acid sequences of non-trivial length. In contrast, strategies with a clonal product of identical DNA molecules, loaded 6

Fig. 5. From sequencing of nucleic acids to alternative informational polymers. (A) Semiconductor sequencing workflow; semiconductor sequencing is based on sensing hydrogen ions released during nucleotide polymerization. (B) The Oxford Nanopore MinIon sequencer enables direct sequencing from double stranded DNA with minimal sample prep, with direct readout via a USB port. C) This nanopore sequencer utilizes phi-29 polymerase or an alternative helicase to strand- displace the DNA base-by-base, yielding controlled movement through the α-hemolysin pore. The sequence is determined by changes in ionic current that are characteristic of the bases near the minimum pore diameter. D) In proposed graphene nanogap sequencing devices, the thin monolayer theoretically enables single-base resolution, with sensing based on how DNA or any appropriately sized polymer modifies the electron tunneling current across the monolayer. E) Demonstrated (solid line) and theoretical (dashed line) routes to sequencing of DNA, RNA, non-standard Xeno Nucleic Acids (XNAs) [7] and other informational polymers. into the sequencing chip, and sequenced on the Ion Torrent diverse types of nucleic acids [7]. In general, a polymer can Personal Genome Machine (PGM). Sequencing is based on be converted to DNA and sequenced, or possibly directly the fundamental chemistry of nucleotide incorporation: one sequenced using a nanopore approach (Fig. 5E). Even type of nucleotide (A,C, G or T) flows past a well; if it without nanopore sequencing, single molecule sequencing matches the next base, polymerase incorporates it, releasing of DNA or RNA is achievable [52], but currently involves a proton (H+). Since this happens on 105 to 106 identical technologies not suitable for in situ use. DNA strands, this results in a pH change that can be sensed by an ion-selective sensor (ISFET) below each well, The ideal technology for Enceladus might be graphene creating a pulse of voltage and yielding a digital signature of nanogap devices, which could permit characterization of the nucleotide sequence. any molecule compatible with the gap size between the graphene electrodes. Varied pore sizes will permit Dramatic simplification may be possible based on nanopore translocation of IPs of different sizes. Due to their low noise sequencing (Fig. 5B-D), which offers the ability to sequence and extreme thinness, graphene monolayers could individual molecules of DNA without amplification. theoretically permit single base recognition of nucleic acids Sequencing of non-standard nucleic acids or more general at high speed, and translocation could be measured as the informational polymers may also be feasible (Fig. 5D) using electron tunneling current across each pore, so that the proposed solid-state nanopores or nanogaps [51] to membrane also acts as the electrode. Damage to the characterize molecules as they flow between a pair of graphene monolayer, such as defects, mechanical damage, electrodes. or radiation damage, will be sensed as a large drop in tunneling current. Other non-standard IPs could potentially It is also possible to use engineered polymerases able to also be sequenced if their sizes are consistent with pore read and write non-standard nucleic acids to detect more geometry. 7

As a backup to the nascent technology of nanopores, we ACKNOWLEDGEMENTS have already demonstrated the ability of PCR and sequencing reagents [53] and miniature non-optical This work was funded under NASA award NNX08AX15G. semiconductor sequencing chips (manuscript submitted to Astrobiology) to survive several analogs of space radiation. REFERENCES The semiconductor sequencing approach could be extended [1] M. P. Callahan, K. E. Smith, H. J. Cleaves, J. Ruzicka, beyond RNA and DNA to several other nucleic acid analogs J. C. Stern, D. P. Glavin, C. H. House, and J. P. using engineered polymerases, although the sample Dworkin, "Carbonaceous meteorites contain a wide preparation requirements are slightly more complicated and range of extraterrestrial nucleobases," Proceedings of it would provide a less general approach to life detection. In the National Academy of Sciences, vol. 108, pp. 13995- addition, semiconductor sequencing and current biological nanopores use biological reagents that are not compatible 13998, Feb 11 2011. with Viking-level sterilization. [2] Z. Martins, O. Botta, M. Fogel, M. Sephton, D. Glavin, J. Watson, J. Dworkin, A. Schwartz, and P. 7. SUMMARY AND CONCLUSIONS Ehrenfreund, "Extraterrestrial nucleobases in the Murchison meteorite," Earth and Planetary Science Searching for life on Enceladus could give us our first Letters, vol. 270, pp. 130-136, Jun 15 2008. glimpse of a second genesis and test whether biochemistry [3] M. Nuevo, S. N. Milam, and S. A. Sandford, is varied or universal. However, some caveats are in order. "Nucleobases and Prebiotic Molecules in Organic First, there may have never been life on Enceladus, or there Residues Produced from the Ultraviolet Photo- may be no life now. Second, the potential subsurface habitat Irradiation of Pyrimidine in NH(3) and H(2)O+NH(3) for life may be periodic – and if so, it may not provide a Ices.," Astrobiology, vol. 12, pp. 295-314, May 2012. long-enough time for life to evolve, or life may not be able [4] M. Nuevo, S. N. Milam, S. A. Sandford, J. E. Elsila, to survive the intervening periods of low heat flux. Third, and J. P. Dworkin, "Formation of uracil from the any life on Enceladus may be present at such a low ultraviolet photo-irradiation of pyrimidine in pure H2O bioburden that it is not feasible to detect it in the plume; the required sample mass may be too high for any reasonable ices.," Astrobiology, vol. 9, pp. 683-695, Sep 2009. orbiter mission and in this case, detection of life on [5] Z. Martins, O. Botta, M. L. Fogel, M. A. Sephton, D. P. Enceladus may have to wait for a lander mission. Glavin, J. S. Watson, J. P. Dworkin, A. W. Schwartz, and P. Ehrenfreund, "Extraterrestrial nucleobases in the Given the presence of detectable informational polymers in Murchison meteorite," Earth and Planetary Science plume particles, there remain several technical hurdles to Letters, vol. 270, pp. 130-136, Jun 15 2008. overcome. Most pressing is the need to determine [6] F. J. Ciesla and S. A. Sandford, "Organic Synthesis via requirements for ice grain capture that yields adequate Irradiation and Warming of Ice Grains in the Solar preservation of informational polymers; this can be Nebula.," Science, Apr 29 2012. experimentally tested using one of several existing research [7] V. B. Pinheiro, A. I. Taylor, C. Cozens, M. Abramov, facilities. Next, simulations and lab tests can be used to M. Renders, S. Zhang, J. C. Chaput, J. Wengel, S.-Y. demonstrate and optimize focusing of charged polymers Peak-Chew, S. H. McLaughlin, P. Herdewijn, and P. under practical mission constraints, which could involve use Holliger, "Synthetic Genetic Polymers Capable of of non-aqueous solvents to facilitate low temperature Heredity and Evolution," Science, vol. 336, pp. 341- operation or reduce power requirements. Finally, while in 344, Feb 20 2012. principle a suitable small sequencer can be built by scaling [8] S. M. Hörst, R. V. Yelle, A. Buch, N. Carrasco, G. existing semiconductor sequencing technologies, this has Cernogora, O. Dutuit, E. Quirico, E. Sciamma-O'Brien, yet to be demonstrated. The first commercial nanopore M. A. Smith, A. Somogyi, C. Szopa, R. Thissen, and V. sequencers may soon address the size, mass, and power Vuitton, "Formation of amino acids and nucleotide constraints for sequencing. bases in a titan atmosphere simulation experiment.," Even given these caveats and challenges, the opportunity at Astrobiology, vol. 12, pp. 809-817, Sep 2012. Enceladus beckons to us: finding life beyond Earth would [9] G. Horneck, D. Stöffler, S. Ott, U. Hornemann, C. S. arguably be the most significant discovery of the space Cockell, R. Moeller, C. Meyer, J.-P. de Vera, J. Fritz, S. exploration endeavor to date. Finding life on Enceladus that Schade, and N. A. Artemieva, "Microbial rock appears to be related to life on Earth would strongly support inhabitants survive hypervelocity impacts on Mars-like panspermia with an origin for life on Earth from the outer host planets: first phase of lithopanspermia planets or beyond. More likely, any life on Enceladus may experimentally tested," Astrobiology, vol. 8, pp. 17-44, represent a second genesis. Because of likely periodic Feb 1 2008. activity on Enceladus, this may provide constraints on how [10] C. Mileikowsky, F. A. Cucinotta, J. W. Wilson, B. fast life can evolve, with implications for the evolution of Gladman, G. Horneck, L. Lindegren, J. Melosh, H. life on Earth. Finally, discovery of a second genesis on Rickman, M. Valtonen, and J. Q. Zheng, "Natural Enceladus would immediately suggest that life is common in the universe. 8

transfer of viable microbes in space.," Icarus, vol. 145, [24] L.-H. Lin, P.-L. Wang, D. Rumble, J. Lippmann-Pipke, pp. 391-427, Jul 2000. E. Boice, L. M. Pratt, B. Sherwood Lollar, E. L. Brodie, [11] H. J. Melosh, "The rocky road to panspermia," Nature, T. C. Hazen, G. L. Andersen, T. Z. DeSantis, D. P. vol. 332, pp. 687-8, Apr 21 1988. Moser, D. Kershaw, and T. C. Onstott, "Long-term [12] O. Mousis, J. I. Lunine, J. H. Waite, and B. Magee, sustainability of a high-energy, low-diversity crustal "Formation Conditions of Enceladus and Origin of Its biome.," Science, vol. 314, pp. 479-482, Oct 20 2006. Methane Reservoir" The Astrophysical Journal Letters, [25] C. D. Parkinson, M. C. Liang, and H. Hartman, vol. 701(1) L39, 2009. "Enceladus: Cassini observations and implications for [13] C. C. Porco, P. Helfenstein, P. C. Thomas, A. P. the search for life," Astronomy and Astrophysics, vol. Ingersoll, J. Wisdom, R. West, G. Neukum, T. Denk, R. 463(1), pp. 353-357, Feb 2007. Wagner, T. Roatsch, S. Kieffer, E. Turtle, A. McEwen, [26] R. H. Tyler, "Ocean tides heat Enceladus," Geophys. T. V. Johnson, J. Rathbun, J. Veverka, D. Wilson, J. Res. Lett., vol. 36, p. L15205, Aug 08 2009. Perry, J. Spitale, A. Brahic, J. A. Burns, A. D. [27] G. W. Ojakangas and D. J. Stevenson, "Episodic Delgenio, L. Dones, C. D. Murray, and S. Squyres, volcanism of tidally heated satellites with application to "Cassini observes the active south pole of Enceladus.," Io," Icarus, vol. 66(2), pp. 341-358, May 1986. Science, vol. 311, pp. 1393-1401, Apr 10 2006. [28] J. H. Roberts and F. Nimmo, "Tidal heating and the [14] B. Giese and C. I. Team, "The topography of long-term stability of a subsurface ocean on Enceladus," EPSC Abstracts, vol. 5, 2010. Enceladus," Icarus, vol. 194, pp. 675-689, 2008. [15] N. Schneider, M. Burger, E. Schaller, M. Brown, R. [29] V. Thór Marteinsson, A. Rúnarsson, A. Stefánsson, T. Johnson, J. Kargel, M. Dougherty, and N. Achilleos, Thorsteinsson, T. Jóhannesson, S. H. Magnússon, E. "No sodium in the vapour plumes of Enceladus," Reynisson, B. Einarsson, N. Wade, H. G. Morrison, and Nature, vol. 459, pp. 1102-1104, Jun 25 2009. E. Gaidos, "Microbial communities in the subglacial [16] F. Postberg, S. Kempf, J. Schmidt, N. Brilliantov, A. waters of the Vatnajökull ice cap, Iceland," The ISME Beinsen, B. Abel, U. Buck, and R. Srama, "Sodium journal, Sep 13 2012. doi: 10.1038/ismej.2012.97 salts in E-ring ice grains from an ocean below the [30] J. M. Hollis, F. J. Lovas, and P. R. Jewell, "Interstellar surface of Enceladus," Nature, vol. 459, pp. 1098-1101, Glycolaldehyde: The First Sugar," The Astrophysical Jun 25 2009. Journal Letters, vol. 540, pp. L107-L110, 2000. [17] F. Postberg, J. Schmidt, J. Hillier, S. Kempf, and R. [31] S. Benner, "Understanding Nucleic Acids Using Srama, "A salt-water reservoir as the source of a Synthetic Chemistry," Acc. Chem. Res., vol. 37, pp. compositionally stratified plume on Enceladus.," 784-797, Oct 19 2004. Nature, vol. 474, pp. 620-622, Jul 30 2011. [32] M. A. Pasek, "Rethinking early Earth phosphorus [18] C. D. Parkinson, M.-C. Liang, Y. L. Yung, and J. L. geochemistry.," Proceedings of the National Academy Kirschivnk, "Habitability of enceladus: planetary of Sciences, vol. 105, pp. 853-858, Feb 22 2008. conditions for life.," Origins of life and evolution of the [33] M. Pasek and D. Lauretta, "Extraterrestrial flux of biosphere, vol. 38, pp. 355-369, Aug 2008. potentially prebiotic C, N, and P to the early Earth.," [19] C. J. A. Howett, J. R. Spencer, J. Pearl, and M. Segura, Origins of life and evolution of the biosphere, vol. 38, "High heat flow from Enceladus' south polar region pp. 5-21, Mar 2008. measured using 10-600 cm-1 Cassini/CIRS data," J. [34] M. A. Pasek and D. S. Lauretta, "Aqueous corrosion of Geophys. Res., vol. 116, p. E03003, Feb 04 2011. phosphide minerals from iron meteorites: a highly [20] D. A. Patthoff and S. A. Kattenhorn, "A fracture history reactive source of prebiotic phosphorus on the surface on Enceladus provides evidence for a global ocean," of the early Earth.," Astrobiology, vol. 5, pp. 515-535, Geophysical Research Letters, vol. 38, L18201, 2011. Aug 2005. [21] C. P. McKay, C. C. Porco, T. Altheide, W. L. Davis, [35] P. Schmitt-Kopplin, Z. Gabelica, R. Gougeon, A. and T. A. Kral, "The possible origin and persistence of Fekete, B. Kanawati, M. Harir, I. Gebefuegi, G. Eckel, life on Enceladus and detection of biomarkers in the and N. Hertkorn, "High molecular diversity of plume.," Astrobiology, vol. 8, pp. 909-919, Oct 2008. extraterrestrial organic matter in Murchison meteorite [22] T. O. STEVENS and J. P. MCKINLEY, revealed 40 years after its fall," Proceedings of the "Lithoautotrophic microbial ecosystems in deep basalt National Academy of Sciences, vol. 107, pp. 2763-2768, aquifers," Science, vol. 270, pp. 450-454, 1995. Feb 16 2010. [23] D. Chivian, E. L. Brodie, E. J. Alm, D. E. Culley, P. S. [36] M. H. Engel and S. A. Macko, "Isotopic evidence for Dehal, T. Z. DeSantis, T. M. Gihring, A. Lapidus, L. extraterrestrial non- racemic amino acids in the Lin, S. R. Lowry, D. P. Moser, P. M. Richardson, G. Murchison meteorite," Nature, vol. 389, pp. 265-268, Southam, G. Wanger, L. M. Pratt, G. L. Andersen, T. 1997. C. Hazen, F. J. Brockman, A. P. Arkin, and T. C. [37] G. Cooper, C. Reed, D. Nguyen, M. Carter, and Y. Onstott, "Environmental Genomics Reveals a Single- Wang, "Detection and formation scenario of citric acid, Species Ecosystem Deep Within Earth," Science, vol. pyruvic acid, and other possible metabolism precursors 322, pp. 275-278, Oct 10 2008. in carbonaceous meteorites," Proceedings of the 9

National Academy of Sciences, vol. 108, pp. 14015- [50] J. M. Rothberg, W. Hinz, T. M. Rearick, J. Schultz, W. 14020, Feb 08 2011. Mileski, M. Davey, J. H. Leamon, K. Johnson, M. J. [38] J.-F. Xiao and J. Yu, "A scenario on the stepwise Milgrew, M. Edwards, J. Hoon, J. F. Simons, D. evolution of the genetic code.," Genomics, proteomics Marran, J. W. Myers, J. F. Davidson, A. Branting, J. R. & bioinformatics, vol. 5, pp. 143-151, Dec 2007. Nobile, B. P. Puc, D. Light, T. A. Clark, M. Huber, J. [39] M. D. Kirnos, I. Y. Khudyakov, N. I. Alexandrushkina, T. Branciforte, I. B. Stoner, S. E. Cawley, M. Lyons, Y. and B. F. Vanyushin, "2-aminoadenine is an adenine Fu, N. Homer, M. Sedova, X. Miao, B. Reed, J. Sabina, substituting for a base in S-2L cyanophage DNA.," E. Feierstein, M. Schorn, M. Alanjary, E. Dimalanta, D. Nature, vol. 270, pp. 369-370, Nov 24 1977. Dressman, R. Kasinskas, T. Sokolsky, J. A. Fidanza, E. [40] P. Tsou, D. E. Brownlee, C. P. McKay, A. D. Anbar, H. Namsaraev, K. J. McKernan, A. Williams, G. T. Roth, Yano, K. Altwegg, L. W. Beegle, R. Dissly, N. J. and J. Bustillo, "An integrated semiconductor device Strange, and I. Kanik, "LIFE: Life Investigation For enabling non-optical genome sequencing.," Nature, vol. Enceladus A Sample Return Mission Concept in Search 475, pp. 348-352, Jul 21 2011. for Evidence of Life.," Astrobiology, vol. 12, pp. 730- [51] H. Postma, "Rapid Sequencing of Individual DNA 742, Aug 2012. Molecules in Graphene Nanogaps," Nano Letters, [41] D. Broemeling, J. Pel, D. Gunn, L. Mai, J. Thompson, 10(2), pp 420-425, Jan 4 2010. H. Poon, and A. Marziali, "An instrument for [52] Y. Kang, M. H. Norris, J. Zarzycki-Siek, W. C. automated purification of nucleic acids from Nierman, S. P. Donachie, and T. T. Hoang, "Transcript contaminated forensic samples," JALA (Charlottesville, amplification from single bacterium for transcriptome Va), vol. 13, pp. 40-48, Feb 1 2008. analysis.," Genome research, vol. 21, pp. 925-935, Jul [42] J. Pel, "A Novel Electrophoretic Mechanism and 2011. Separation Parameter for Selective Nucleic Acid Concentration Based on Synchronous Coefficient of [53] C. E. Carr, H. Rowedder, C. Vafadari, C. S. Lui, E. Drag Alteration (SCODA)," University of British Cascio, M. T. Zuber, and G. Ruvkun, "Radiation Columbia, 2009. resistance of biological reagents for in-situ life [43] T. Noguchi, T. Nakamura, K. Okudaira, H. Yano, S. detection," Astrobiology, vol. 13, no. 1 (in press), 2013. Sugita, and M. J. Burchell, "Thermal alteration of hydrated minerals during hypervelocity capture to silica BIOGRAPHY aerogel at the flyby speed of Stardust," Meteoritics & Planetary Science, vol. 42, pp. 357-372, Apr 2007. Christopher. E. Carr is a Research Scientist in the MIT Department of [44] M. J. Burchell, M. J. Cole, J. A. M. McDonnell, and J. Earth, Atmospheric and Planetary C. Zarnecki, "Hypervelocity impact studies using the 2 Sciences, and a Research Fellow in MV Van de Graaff accelerator and two-stage light gas the Massachusetts General Hospital gun of the University of Kent at Canterbury," Department of Molecular Biology. Measurement Science and Technology, vol. 10, pp. 41- Dr. Carr is the science PI for a 50, Feb 01 1999. NASA-funded life detection [45] M. J. Burchell, M. J. Cole, M. C. Price, and A. T. instrument based on nucleic acid Kearsley, "Ice Impacts on Aerogel and Stardust Al sequencing, the Search for Extra- Foil," Meteoritics and Planetary Science Supplement, Terrestrial Genomes (SETG). He received dual B.S. vol. 74, p. 5345, Sep 01 2011. degrees in astronautics and electrical engineering from [46] T. Han, Y. Nazarenko, P. J. Lioy, and G. Mainelis, MIT, worked on Mars Sample Return at JPL, and "Collection efficiencies of an electrostatic sampler with completed his doctoral work in the Harvard-MIT Division superhydrophobic surface for fungal bioaerosols.," of Health-Sciences and Technology. Indoor air, vol. 21, pp. 110-120, May 2011. [47] M. F. Mora, A. M. Stockton, and P. A. Willis, Maria T. Zuber is the E. A. "Microchip capillary electrophoresis instrumentation Griswold Professor of Geophysics in for in situ analysis in the search for extraterrestrial the MIT Department of Earth, life.," ELECTROPHORESIS, vol. 33, pp. 2624-2638, Atmospheric and Planetary Sep 2012. Sciences. Dr. Zuber has been [48] X. Gao, B.-H. Tan, R. J. Sugrue, and K. Tang, "MALDI involved in ten NASA planetary Mass Spectrometry for Nucleic Acid Analysis.," Topics missions aimed at mapping the in current chemistry, Sep 28 2012. Moon, Mars, Mercury, and several [49] R. Stepanauskas, "Single cell genomics: an individual asteroids. She received her B.A. in astrophysics from the University of look at microbes.," Current Opinion in Microbiology, Pennsylvania and Sc.M. and Ph.D. in geophysics from 15(5):613-20, Oct 2012. doi: Brown University. She was on the faculty at Johns 10.1016/j.mib.2012.09.001. Hopkins University and served as a research scientist at

10 the NASA Goddard Space Flight Center in Maryland. Most recently she served as the principal investigator for the Gravity Recovery and Interior Laboratory (GRAIL) mission; twin spacecraft in lunar orbit provided high- resolution gravity field mapping of the Moon.

Gary Ruvkun is a molecular biologist and professor at the Masschusetts General Hospital (MGH) and Harvard Medical School, and an associate member of the Broad Institute. Dr. Ruvkun is a member of the National Academy of Sciences and the National Research Council Committee on Planetary Science and Astrobiology. Dr. Ruvkun originated the SETG project and has long been involved in studies of RNA biology, microbial evolution and diversity. He is most well known for co-discovering micro-RNAs, short RNAs that regulate gene expression, mainly in eukaryotic cells.