978-1-4673-1813-6/13/$31.00 ©2013 IEEE 1

Life Detection with the Enceladus Orbiting Sequencer Christopher E. Carr & Maria T. Zuber

Massachusetts Institute of Technology, Department of Earth, Atmospheric and Planetary Sciences

Cambridge, MA 02139 chrisc@mit.edu, zuber@mit.edu

Gary Ruvkun Massachusetts General Hospital, Dept. of Mol. Biology

Harvard Medical School, Dept. of Genetics Boston, MA 02114

ruvkun@molbio.mgh.harvard.edu

Abstract— Widespread organic synthesis in the early solar nebula led to delivery of similar complex organics, probably including nucleobases or their precursors, to many potentially habitable locations such as Mars, Europa, and Enceladus. If life evolved beyond Earth, the presence of these organics could have biased life towards utilization of informational polymers (IPs) like RNA or DNA. Given this, searching for and sequencing any such IPs offers a definitive, information rich, 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. Recent discovery of a salt-water plume likely derived from sub-surface liquid reservoirs provides direct access to this potentially habitable environment. We describe an instrument concept, the Enceladus Orbiting Sequencer (EOS), specifically geared to search for life on Enceladus. As a payload on board an Enceladus flyby or orbiter mission, EOS would capture ice grains from the plume, then concentrate and characterize any long charged polymers using nanopore or semiconductor sequencing. Searching for life on Enceladus could give us our first glimpse of a second genesis and test whether biochemistry is varied or universal.

TABLE OF CONTENTS

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 beyond Earth could be based on nucleic acid-like polymers such as RNA or DNA. Furthermore, complex organic synthesis is widespread in stellar nebulae [3, 6], likely around all stars. As a result, common sources of organic material were delivered to multiple potentially habitable zones in our solar system, such as Mars, Europa, and Enceladus. This delivery could have biased the development of life towards similar biochemical solutions such as utilization of informational polymers (IPs) like RNA and DNA. An even stronger case can be made to search for

RNA or DNA on Mars due to the significant meteoritic transfer between Earth and Mars [1, 9-11]. However, the ability to detect and sequence other nucleic acids and more generic IPs would provide additional sensitivity. We review prospects for extant life on Enceladus and describe an instrument concept specifically geared to searching for life on this remarkable icy moon.

2. IS ENCELADUS HABITABLE? A Brief History of Enceladus

Enceladus is a volatile-rich (density 1.6 g/cm3) moon located in Saturn’s E-ring (Fig. 1A). Enceladus likely formed from icy planetesimals produced in the solar nebula that were then partially devolatilized during their consolidation in the Saturn subnebula [12]. Its icy shell may cover a subsurface ocean, which may be in direct contact with its rocky core (Fig. 1B). Plume ice from Enceladus feeds the Saturn E-ring, which has been observed to be stable since the 1960s. As Enceladus sweeps counter-clockwise around Saturn and through the ring, it collects irradiated ring particles and leaves a shadow (Fig. 1C).

Fig. 1. Saturn's icy moon Enceladus. (A) Ice from

Enceladus forms Saturn's outermost ring. (B) Enceladus has a rocky core of radius 150-160 km, covered by 90-100 km of H2O. (C) Enceladus both creates and sweeps through the E-ring. The bright dot is the plume, whereas the moon is a tiny black region above the bright dot. (D) The surface of

Enceladus has geologically young, fractured (blue) regions and older, cratered regions. (E) Cassini identified the

Enceladus plume during a 2005 flyby. All images from NASA Cassini mission (CICLOPS); panel A is contrast

enhanced, B is artist's impression, D and E are false color.

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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

Imaging of the SPT revealed four main subparallel furrows and ridges (sulci), each about 130 km in length with central two km wide depressions. These sulci are associated with heat anomalies and eight known plume sources (Fig. 2A-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 (CDA) revealed a population of sodium-rich (0.5-2%) grains in the plume, consistent with a subsurface ocean [16] and an explanation for the lack of salt in the plume vapor: sodium salts would remain in the liquid phase as the water freezes out as part of the ice crust, with sublimation of this crust yielding plume gas. Later flybys and analysis by the CDA strengthened the case for a subsurface salt-water reservoir [17], consistent with models of a subsurface ocean in contact with the moon’s rocky core (Fig. 2C).

To date, observations and models argue for three general types of ice grains in the plume [17]: Type I grains are small, salt poor, and likely formed from nucleation of gas phase water vapor, leading to high ejection speeds and explaining their dominant contribution to the E-ring. Type II grains contain organic and/or siliceous material, might indicate surface/ocean communication, and, speculatively, could be derived from solid clathrates [18]. More organics are observed in the center of the plume [17]. Type III grains are salt-rich larger particles found in higher densities in the lower part of the plume, and presumably formed from a rapidly frozen spray of salt water. They constitute 70% of the grains above 0.2 μm in size, have lower ejection speeds, and dominate the mass flux (99%).

Extant Life?

If extant life exists on Enceladus, it either evolved there (a second genesis) or was carried there from somewhere else (panspermia). Either way, life as we know it requires liquid water, essential nutrients (mainly made of the CHNOPS elements), and a source of energy (chemical or redox gradient). If we are to posit a second genesis on Enceladus, it must also have had time to evolve. We address these considerations in turn.

Panspermia?—Extremely limited meteoritic transfer between the inner planets (including Earth) and Enceladus virtually guarantees that any life on Enceladus will not be

related to life on Earth, unless life on Earth and Enceladus both derived from another (outer planets or beyond) source that had significant meteoritic transfer to both bodies.

Liquid water?—Despite the generally frigid surface temperature (-200˚C) of Enceladus, several plume and surface characteristics argue for a liquid water source for at least part of the plume. First, the substantial heat output of the SPT, at 16 GW [19], is most consistent with an elevated temperature source. Second, modeling of plume ice particle growth suggests source temperatures of at least 190K [18]. Third, the presence of sodium ice grains requires rapid freezing, consistent with quickly cooled liquid water. Fourth, the fracture patterns in the south polar terrain are best explained by a global liquid ocean [20]. Of note, methanogens in permafrost can stay metabolically active at -20˚C by utilizing tightly bound unfrozen water [21].

Essential nutrients?—Enceladus acquired organic material as a consequence of its primordial origin, and by delivery of organics by comets and meteors. Some organic material has been definitively detected in the plume [17]. The organics available to any origin of life on Enceladus may have even included the building blocks of life as we know it (see Why search for nucleic acids and related polymers? below).

Energy source? — Any life at Enceladus is unlikely to use photosynthesis due to low light levels and the inability of light to penetrate into the subsurface. Similarly, oxidative phosphorylation appears unlikely. The most likely model for any life on Enceladus is chemolithotrophy.

Ecosystems that apply to Enceladus (depending neither on photosynthesis nor on oxygen) include methanogens that consume H2 produced by serpentinization (iron oxidation by water) of olivine in the Columbia River basalts [22], and sulfur-reducing bacteria that utilize H2 generated as a byproduct of radioactive decay [23, 24] generating H2S. Deuterium-to-hydrogen (D/H) ratio measurements may help determine if the source of methane in the plume is derived

Fig. 2. Plume sources and likely structure. (A) The four major sulci are associated with eight plume sources (yellow points) and large heat anomalies (overlay). (B) Damascus Sulcus, relief 10x exaggerated. (C) Plume source model: I, II, III indicate predominant ice grain type. Image credits

NASA/JPL/GSFC/SwRI/SSI for A and B.

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from serpentinization or is primordial in nature [12]. McKay et al. [21] also describe a hypothetical methane cycle on Enceladus in which methanogens consume H2 and the CH4 is thermally processed in hot rock to H2. They also suggest that a very low ratio (0.001) of non-methane hydrocarbons to CH4 may indicate a biological CH4 source.

Some have argued that the biggest redox limitation for Enceladus is a lack of oxidants [21]. However, at least two potential redox cycles are available: turnover with the surface, bringing a supply of oxidants to a sub-surface ocean, and thermal reformation of chemicals at depth (described above).

While Europa’s surface ice is heavily irradiated, radiation levels at Enceladus are much lower, limiting the available oxidants at the surface. However, the E-ring may act as a chemical processor [25], providing an extended surface to generate oxidants that are later collected by Enceladus. Turnover at active sites could bring surface oxidants into contact with a sub-surface ocean and help maintain redox gradients.

Enough time to evolve life?—Most current models of the moon’s energy budget cannot explain the large heat anomaly associated with the plume. For example, radiogenic heating can explain less than 1 GW. One possibility that does explain the heat anomaly is that of global tides from a shallow (<10 km) ocean [26]. Another possibility is that the current heat output of Enceladus may be above the steady state value. How can this be explained?

Enceladus may undergo periodic oscillations in surface heat flow due to changes in orbital eccentricity, just as does Io [27]. Io is predicted to have peak surface heatflow that lags changes in eccentricity. While the current heatflow on Enceladus seems unsustainable in comparison to its current orbital eccentricity (0.005), this could be explained by past higher eccentricity [21]. This raises important questions: If activity on Enceladus is periodic, how long does it last? One hint is that it may take 30 million years to freeze out an Enceladus subsurface ocean [21, 28]. If Enceladus is habitable during periods of high heat flow, could life survive during periods of reduced activity? Or could it evolve during one period of activity?

Searching for life on Earth in isolated subglacial lakes may inform our search for life on Enceladus. Preliminary results from the Russian drilling expedition that has drilled through 3.5 km of ice into Lake Vostok, a sub-glacial Antarctic lake thought to be isolated for millions of years, has not yet found cells above the background level in their clean room (10 microbes/ml); most of the cells have been identified as contaminants from drilling oil (Nature, Brian Owens, 18 Oct 2012). Microbes have been identified in other isolated subglacial lakes [29], where they are thought to have migrated from the subsurface.

Just as we can apply exquisitely sensitive molecular biology techniques to the detection of life in these environments, so

we may be able to apply them to detection of life on Enceladus.

Why search for nucleic acids and related polymers?

All known life utilizes deoxyribonucleic acid (DNA, Fig. 3A) or ribonucleic acid (RNA, Fig. 3B), a linear polymer made from nucleotides, each one composed of a backbone molecule (phosphate group), a nucleobase, and a tripartite group (sugar) linking the base and adjacent backbone molecules. The pattern of nucleobases directly encodes the enzymatic activity of RNAs and indirectly encodes the amino acid sequence of proteins through the genetic code, which maps triplet codons to a smaller group of amino acids.

Nucleotide structure is likely a consequence of common building blocks: Presence of the ribose precursor glycolaldehyde in interstellar space [30] could have led to the development of IPs based on glycol (GNA), threose (TNA), or ribose (RNA/DNA) sugars (Fig. 3C). Charged backbones are likely universal for aqueous life [31]: they separate the physical properties of the polymer from its associated information content, facilitating replication and evolution. This places limits on possible backbones other than phosphate. In addition, meteorites delivered phosphide minerals to habitable environments, including Earth [32-34], where they may have supplemented a meager terrestrial supply of phosphorus. But what about the third nucleotide component: Might life beyond Earth use non-standard nucleobases? Current evidence suggests perhaps not.

Widespread synthesis of complex organics occurred early in the history of the solar system in the solar nebula [6]. Lab experiments (Fig. 3D) reveal the formation of amino acids and nucleobases [3, 4] including adenine (A), cytosine (C), guanine (G), and thymine (T) (the standard bases in DNA, which form A-T and C-G base pairs through hydrogen bonding) and uracil (U) (which substitutes for T in RNA). Thus, widely mixed in the nebula, the building blocks of life as we know it were likely delivered to all potentially habitable zones in the solar system via comet and meteor impacts. Analysis of meteorites (Fig. 3D, blue bars) has confirmed the presence of extraterrestrial amino acids and nucleobases [1, 5, 35-37] including G, hypoxanthine, xanthine, A, purine, and diaminopurine [1].

Non-standard bases play important roles in life today. For example, hypoxanthine is part of the inosine (I) nucleoside (a nucleobase linked to a sugar backbone) found in transfer RNAs, where it is essential for proper translation of the genetic code (from 64 base triplet codons to 21 amino acids) in wobble base pairs through promiscuous pairing with A, C, or U [38]. Thus, the error-correcting capabilities of the genetic code may be enabled by extraterrestrial chemistry.

Another example is xanthine, which is found in many human tissues and plays a role in generation of free radicals. In addition, diaminopurine base pairs with both xanthine and T (substituting for A and leading to increased basepair

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stability; cyanophage S-2L uses this property to facilitate host evasion [39]). Thus, these nonstandard base pairs are not foreign to biology, but are in fact used by it today.

Further synthesis took place in reducing planetary atmospheres (early Earth, Mars, Venus and present-day Titan). For example, Cassini measurements have identified unknown large organic molecules in Titan’s upper atmosphere; lab-based experiments (Fig. 3D, red bars) yielded 18 molecules corresponding to amino acids and all of the standard nucleobases [8]. Thus, the basic building blocks of life as we know it may form in reducing planetary atmospheres, in interstellar space, or, as has long been known, through aqueous chemistry. Finally, the availability of common building blocks in multiple habitable zones may have biased “independent” genesis events towards the use of similar informational polymers (IPs).

3. MISSION ARCHITECTURE

Unlike Mars and Europa, brine from the interior of Enceladus can be acquired while orbiting or possibly during a flyby, due to the geysers erupting from its surface. Like Mars, but unlike Europa, Enceladus also presents a moderate radiation environment without extensive ionized trapped particles, a benefit to radiation-sensitive instrumentation. Enceladus also enables us to access the planetary interior without a high risk of forward contamination [18]. This is unlike Mars, where potentially habitable environments would need to be directly accessed

from the surface most likely using a drill. Similarly, accessing habitable zones on Europa may require drilling through an unknown layer of ice to reach the subsurface ocean. Thus, life detection missions on Mars and Europa are not only technically complicated but impose a higher risk of forward contamination of potentially habitable zones. Enceladus flyby missions offer a variety of end-of-life options, while orbiter missions could deorbit onto Enceladus (with obvious planetary protection issues) or leave orbit using minimal delta-v and exercise other options.

Key trades: Flyby or Orbiter? In-situ vs. Return?

A key trade that must be pursued is to determine whether volatiles, and in particular, intact nucleic acids or related informational polymers, can be recovered from plume particles at sample capture velocities consistent with flyby 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.

Another key trade is whether to carry out extensive in-situ analysis or carry out a sample return mission. The baseline cost of a sample return mission may make it prohibitively more expensive. In addition, a sample return mission to Enceladus would likely be classified as planetary protection category V (restricted), due to the potential for chemical evolution and/or life on Enceladus. Under this categorization, the cost associated with planetary protection could potentially exceed the baseline sample return mission cost [40]. In addition, during the return trip of up to 5 years

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).

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in one proposal [40], samples would be exposed to space radiation and would otherwise need to be preserved under pristine conditions.

Thus, even aside from the planetary protection-associated costs, we argue for at least some in-situ analysis, to facilitate use of fresh samples and to reduce the risks of contaminating the samples with Earth organics.

Plume Sampling

Several post-Cassini follow-up missions to study Enceladus have been proposed. Two (Titan and Enceladus $1B Mission Feasibility Study and the Enceladus Flagship Mission Concept Study, reviewed by Tsou et al. [40]) have extremely high sample capture speeds of 7 to 10 km/s, even higher than the Stardust mission (6.1 km/s). The slowest Cassini relative velocity to date was 6.2 km/s (flyby E-13 on December 21, 2010, out of 19 total flybys through October 2012). Recently, Tsou et al. [40] proposed a trajectory that could achieve Enceladus flybys at reduced speeds, as low as 3 to 4.5 km/s. Plume velocities could modify the actual sample capture velocities, but many of the particles can be sampled in a stagnation region before they fall back to the surface of Enceladus. Therefore the plume velocity can be ignored to first order.

Once in orbit, sampling could be accomplished at much lower speeds and higher sampling rates. For example, at an 80 km orbit, orbital velocity is only 147 m/s, and the sampling frequency could be as high as 6 times per day instead of Cassini’s average of a few times a year.

For either an orbiter or flyby mission, orientation control would be important to align the desired ram direction with the particle flux, which is approximately 1 ice particle per cubic meter at ~80 km orbit [40]. Lower orbits may be required to acquire adequate materials for in-situ analysis 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-based aerogel (SEAgel, US patents 5,382,285 and 5,360,828) can be made with densities as low as 1.5 mg/cm3 (versus 1.25 mg/cm3 for air). Tsou et al. [40], in their proposed LIFE Enceladus sample return mission, would

decrease aerogel density down to 2 mg/cm3 to facilitate less destructive sample capture, and collect and secure volatiles.

We propose to re-seal compartments to protect against volatile loss and possibly allow multiple sampling events for a single compartment. We assume that the shock pressures involved will be enough to disrupt any cells in plume particles to enable later release of genetic material (informational polymers, IPs). This assumption should be tested experimentally. Additional lysis steps can be taken at the cost of greater complexity.

Once ice grains have been collected, any IPs must be greatly concentrated to cope with extremely low sample volume and facilitate low downstream volumes. The top candidate for this is Synchronous Coefficient of Drag Alteration (SCODA) [41, 42], which can achieve concentration up to 5000X, while also demonstrating extreme contaminant rejection (Fig. 4B). On a related note, the Enceladus Amino Acid Sampler, a NASA ASTID-funded project, will attempt to demonstrate a 104 concentration factor.

Concentration of nucleic acids via SCODA relies upon the physical properties of long polymer chains, and specifically upon their inability to quickly reorient after directional changes in the electric field. Four electrodes are used to apply rotating dipole and quadruple electric fields to generate a divergent velocity field, allowing for selective concentration of long charged polymers at the geometric center of the electrodes when the time-averaged electric field is zero. While long charged polymers focus, other charged or neutral particles do not, yielding selective focusing of polymers like, but not limited to, RNA or DNA. For DNA, polymers longer than 200 to 300 bp are focused, equivalent to a few times the persistence length. SCODA has been commercialized by Boreal Genomics and has demonstrated high efficiency in low abundance samples.

Fig. 4. EOS sample acquisition. (A) Ice grains decelerate through aerogel and deposit into agarose. (B) SCODA [41,

42] permits focusing of generic charged polymers to achieve massive sample concentration and contaminant rejection.

Image credit: Boreal Genomics.

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Once the informational polymers have been focused, they can be characterized through sequencing. We now discuss issues relating to ice grain capture and then discuss approaches to sequencing of informational polymers.

5. ICE GRAIN CAPTURE

The ice grains emitted by the geysers (up to 100 μm diameter) are large enough to harbor microbial cells (2 μm typical) and the largest grains are negatively charged and carry 90% of electrons in the plume. Collecting these impacting grains while leaving any IPs adequately intact may pose the biggest challenge.

The temperature rise as a function of velocity clearly favors an Enceladus orbiter. For example, at the Stardust collection speed (6.1 m/s), the temperature rise for an ice grain would come to nearly 2 x 104 K (assuming all kinetic energy is spread evenly over the ice grain and using the specific heat of water). Reducing the velocity to 200 m/s gives a more moderate 20 K. In practice, much higher velocities may be possible due to uneven heating of grains: gradients as large as 2500 K/μm have been estimated for experimental studies of hypervelocity capture of meteorite powders by aerogel [43]. Ice grains are unlikely to support gradients this large but may still undergo extremely uneven heating.

Experimental testing of ice grain capture is clearly required. Some Stardust aerogel tracks end without dust particles, indicating potential loss of volatile material. Recently, a two-stage light-gas gun [44] has been used to study whether ice grains could be captured by aerogel at stardust velocities of 6.1 km/s [45]. Analysis of impact tracks suggested no terminal grains, consistent with loss of volatile material. Tests at lower velocities such as 2 to 4.5 km/s should be carried out to ascertain whether capture of relatively intact ice grains is feasible for an Enceladus flyby mission.

For an orbiter mission, it may be possible to use electrostatic focusing of large negatively charged grains to dramatically increase the collection efficiency and focusing of grains. Of note is recent progress in this area for airborne particle collection [46].

6. INFORMATIONAL POLYMER SEQUENCING

Why sequencing?

If life beyond Earth uses nucleic acids, either because it is related or because such life evolved from common precursors to use similar informational molecules, we can leverage the powerful tools that have emerged from billions of dollars invested in genomics that are now found in thousands of labs and will soon be ubiquitous in health care and beyond.

Sequencing is sensitive down to a single molecule and is highly specific: there are no known abiotic routes to nucleic acid sequences of non-trivial length. In contrast, strategies

that seek to avoid the assumption that life elsewhere may share any particular features with life on Earth are not as sensitive, nor do they provide definitive answers. Two examples include mass spectrometry or microcapillary electrophoresis with laser induced fluorescence [47].

Other approaches?

Mass spectrometry (MS) can be used for sequencing of proteins or nucleic acids, but is generally limited to lengths of less than 30 base pairs (bp) [48]. In addition, MS is not particularly sensitive and is thus limited to identifying molecules of relative abundance. MS was studied intensively for DNA sequencing during the human genome project and rejected in favor of more successful alternatives. MS has been more successful in protein sequencing, due to the tendency of nucleic acid polymers to fragment or undergo base loss during ionization. Thus, mass spectrometry is not currently a viable option for in situ sequencing of non-trivial length nucleic acid polymers, particularly when abundances are low.

Microcapillary electrophoresis with laser induced fluorescence (μCE-LIF) is a sensitive (parts per trillion, ppt) way to identify amino acids, nucleobases, and other molecules, and is being developed for in situ use [47]. The target molecules are extracted and labeled with a fluorescent probe, then electrophoresis is used to separate the targets by size. μCF-LIF could be used with Sanger sequencing to separate labeled DNA fragments, generating one sequence per separation. While sensitive, μCE-LIF is less sensitive than sequencing and cannot efficiently yield large numbers of reads. In contrast, sequencing can generate a nearly complete genome from a single cell [49]. If a cell has a single-copy 5 Mb genome (5 femtograms DNA) and is captured into 1 μl of water, detection corresponds to 5 parts per trillion sensitivity. The actual sensitivity is higher, because sequencing of this cell will involve ~1 M independent sequencing reads.

Sequencing nucleic acids and related polymers

Until recently, sequencing instruments have been large, heavy, complex, and required specialized reagents and optics. However, it is now possible to consider in-situ massively parallel metagenomic sequencing on a commercially produced Ion Torrent solid-state chip [50]. This complementary metal-oxide semiconductor (CMOS) chip is similar to those found in digital cameras, but instead of capturing light, it detects pH changes resulting from addition of bases to growing DNA chains. Current generation chips have from 1.2 M to 165 M wells. This enables concurrent sequencing in millions of wells in parallel, requires no imaging or optics, and is extremely small, fast, and robust.

To use this semiconductor sequencing chip (Fig. 5A), adaptors are ligated to end-repaired fragmented DNA to make a sequencing library, which is amplified through emulsion PCR and enriched to give positive beads, each with a clonal product of identical DNA molecules, loaded

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into the sequencing chip, and sequenced on the Ion Torrent Personal Genome Machine (PGM). Sequencing is based on the fundamental chemistry of nucleotide incorporation: one type of nucleotide (A,C, G or T) flows past a well; if it matches the next base, polymerase incorporates it, releasing a proton (H+). Since this happens on 105 to 106 identical DNA strands, this results in a pH change that can be sensed by an ion-selective sensor (ISFET) below each well, creating a pulse of voltage and yielding a digital signature of the nucleotide sequence.

Dramatic simplification may be possible based on nanopore sequencing (Fig. 5B-D), which offers the ability to sequence individual molecules of DNA without amplification. Sequencing of non-standard nucleic acids or more general informational polymers may also be feasible (Fig. 5D) using proposed solid-state nanopores or nanogaps [51] to characterize molecules as they flow between a pair of electrodes.

It is also possible to use engineered polymerases able to read and write non-standard nucleic acids to detect more

diverse types of nucleic acids [7]. In general, a polymer can be converted to DNA and sequenced, or possibly directly sequenced using a nanopore approach (Fig. 5E). Even without nanopore sequencing, single molecule sequencing of DNA or RNA is achievable [52], but currently involves technologies not suitable for in situ use.

The ideal technology for Enceladus might be graphene nanogap devices, which could permit characterization of any molecule compatible with the gap size between the graphene electrodes. Varied pore sizes will permit translocation of IPs of different sizes. Due to their low noise and extreme thinness, graphene monolayers could theoretically permit single base recognition of nucleic acids at high speed, and translocation could be measured as the electron tunneling current across each pore, so that the membrane also acts as the electrode. Damage to the graphene monolayer, such as defects, mechanical damage, or radiation damage, will be sensed as a large drop in tunneling current. Other non-standard IPs could potentially also be sequenced if their sizes are consistent with pore geometry.

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.

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As a backup to the nascent technology of nanopores, we have already demonstrated the ability of PCR and sequencing reagents [53] and miniature non-optical semiconductor sequencing chips (manuscript submitted to Astrobiology) to survive several analogs of space radiation. The semiconductor sequencing approach could be extended beyond RNA and DNA to several other nucleic acid analogs using engineered polymerases, although the sample preparation requirements are slightly more complicated and it would provide a less general approach to life detection. In addition, semiconductor sequencing and current biological nanopores use biological reagents that are not compatible with Viking-level sterilization.

7. SUMMARY AND CONCLUSIONS Searching for life on Enceladus could give us our first glimpse of a second genesis and test whether biochemistry is varied or universal. However, some caveats are in order. First, there may have never been life on Enceladus, or there may be no life now. Second, the potential subsurface habitat for life may be periodic – and if so, it may not provide a long-enough time for life to evolve, or life may not be able to survive the intervening periods of low heat flux. Third, any life on Enceladus may be present at such a low bioburden that it is not feasible to detect it in the plume; the required sample mass may be too high for any reasonable orbiter mission and in this case, detection of life on Enceladus may have to wait for a lander mission.

Given the presence of detectable informational polymers in plume particles, there remain several technical hurdles to overcome. Most pressing is the need to determine requirements for ice grain capture that yields adequate preservation of informational polymers; this can be experimentally tested using one of several existing research facilities. Next, simulations and lab tests can be used to demonstrate and optimize focusing of charged polymers under practical mission constraints, which could involve use of non-aqueous solvents to facilitate low temperature operation or reduce power requirements. Finally, while in principle a suitable small sequencer can be built by scaling existing semiconductor sequencing technologies, this has yet to be demonstrated. The first commercial nanopore sequencers may soon address the size, mass, and power constraints for sequencing.

Even given these caveats and challenges, the opportunity at Enceladus beckons to us: finding life beyond Earth would arguably be the most significant discovery of the space exploration endeavor to date. Finding life on Enceladus that appears to be related to life on Earth would strongly support panspermia with an origin for life on Earth from the outer planets or beyond. More likely, any life on Enceladus may represent a second genesis. Because of likely periodic activity on Enceladus, this may provide constraints on how fast life can evolve, with implications for the evolution of life on Earth. Finally, discovery of a second genesis on Enceladus would immediately suggest that life is common in the universe.

ACKNOWLEDGEMENTS This work was funded under NASA award NNX08AX15G.

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[53] C. E. Carr, H. Rowedder, C. Vafadari, C. S. Lui, E. Cascio, M. T. Zuber, and G. Ruvkun, "Radiation resistance of biological reagents for in-situ life detection," Astrobiology, vol. 13, no. 1 (in press), 2013.

BIOGRAPHY Christopher. E. Carr is a Research Scientist in the MIT Department of Earth, Atmospheric and Planetary Sciences, and a Research Fellow in the Massachusetts General Hospital Department of Molecular Biology. Dr. Carr is the science PI for a NASA-funded life detection instrument based on nucleic acid sequencing, the Search for Extra-

Terrestrial Genomes (SETG). He received dual B.S. degrees in astronautics and electrical engineering from MIT, worked on Mars Sample Return at JPL, and completed his doctoral work in the Harvard-MIT Division of Health-Sciences and Technology.

Maria T. Zuber is the E. A. Griswold Professor of Geophysics in the MIT Department of Earth, Atmospheric and Planetary Sciences. Dr. Zuber has been involved in ten NASA planetary missions aimed at mapping the Moon, Mars, Mercury, and several asteroids. She received her B.A. in astrophysics from the University of

Pennsylvania and Sc.M. and Ph.D. in geophysics from Brown University. She was on the faculty at Johns Hopkins University and served as a research scientist at

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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.

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