BEYOND RNA AND DNA: IN-SITU SEQUENCING OF INFORMATIONAL POLYMERS. C. E. Carr1-3,*, G. Ruvkun2-3 and M. T. Zuber1. 1Department of Earth, Atmospheric and Planetary Sciences, MIT, Cambridge MA. 2Department of Molecular Biology, Massachusetts General Hospital, Boston, MA. 3Department of Genetics, Har-vard Medical School, Boston, MA. *Correspondence to chrisc@mit.edu.

Introduction: Nucleic acid sequencing provides a

powerful approach to search for life beyond Earth, as well as to monitor levels of forward contamination [1, 2]. A strong case can be made to look for RNA or DNA-based life on Mars [3, 4]: any life there could have a common ancestor with life on Earth due to ex-tensive meteoritic transfer between our two planets [5-9] and the potential for transfer of viable microbes.

Here we argue that in-situ sequencing has a role in life detection even beyond the context of meteoritic transfer, such as searching for life in potentially habit-able environments on Enceladus [10, 11], or Europa [12]. This is because 1) sequencing of non standard nucleic acids is now possible, and 2) it may also be possible to sequence even more generic informational polymers (IPs). Semiconductor sequencing and the imminent arrival of nanopore sequencing may facili-tate in-situ sequencing of RNA, DNA, and other so-called xeno nucleic acid (XNA) polymers. Semicon-ductor sequencing is reliable and its reagents and chips survive analogs of space radiation consistent with a two-year Mars mission. In contrast, nanopore sequenc-ing is unproven but offers lower bias, simplicity, and the possibility to directly sequence XNAs and perhaps even more general IPs.

Targeting IPs beyond RNA and DNA may yield high sensitivity and specificity without assuming that any life elsewhere uses precisely the same IPs as life on Earth. Discovery of such IPs and their sequences would reveal the extent to which life on Earth and elsewhere shares a common ancestry or biochemistry.

Why search for RNA/DNA? Sequencing RNA or DNA is a powerful approach to characterize life as we know it, and may be able to detect life beyond Earth also based on these IPs. Significant meteoritic ex-change, such as has occurred between Mars and Earth [5-9], would increase the probability that any life in potential habitable zones would utilize similar or iden-tical IPs. Thus, sequencing of RNA or DNA may be able to detect life under scenarios of shared ancestry.

Why look for nucleic acids beyond Mars? Com-plex organics including nucleobases or their precursors are found in meteorites and cometary samples [13-17] and in interstellar space [18]. A major source of these organics may be irradiation-induced organic synthesis in stellar nebulae [19], a process that may be common to most stars (Figure 1).

Figure 1. Delivery of similar organic material to mul-tiple habitable zones: if life arose beyond Earth, what informational polymers would it use? As a result, similar organic material may be delivered to multiple habitable zones within a given solar sys-tem. This may bias the evolution of life towards utili-zation of a common set of precursor molecules. Thus, if life arose in multiple habitable zones, it may utilize similar informational polymers (IPs), even in the ab-sence of meteoritic exchange.

When might the search for RNA or DNA fail? Life on Mars, if it developed, could have evolved to utilize a different IP; however, any Earth-related life there might have retained the ancestral IP. If such an-cestrally-related life first evolved to utilize RNA, life on Mars might be stuck in the RNA world and offer a snapshot of Earth’s deep past. Due to limited meteorit-ic exchange, targeting of RNA or DNA would be more likely to fail in potential habitable environments such as the probable liquid water oceans beneath Europa [12], Enceladus [10, 11] and possibly Titan [20].

Alternatives to RNA/DNA: Although it is possi-ble that life beyond Earth could utilize RNA or DNA, life elsewhere could utilize a different polymer such as TNA [21, 22] or GNA [23], which have been proposed as possible precursors. For example, life might evolve based on an IP that reflects the availability of endemic or delivered organic material or environmental condi-tions that make a specific polymer more advantageous (tolerance to pH, salt, temperature, replication fidelity, and other characteristics). In this case, different origins of life could be associated with different IPs. Another possibility is that different origins may have utilized a similar “ancestral” IP but evolved along alternative paths, displacing the “ancestral” IP with new polymers, or retaining the “ancestral” IP.

Complex organics produced around

all stars Delivered to multiple potentially

habitable zones

Earth

Mars

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Dust, Comets,Meteors GNA/TNA?? RNA DNA

Meteoritic ExchangeCommon ancestor / 2nd genesis / no life

2nd genesis / no life

2nd genesis / no life

2nd genesis / no life

Life evolved one or more times

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Beyond RNA and DNA: A potentially more sensitive approach is to enable sequencing of a more broad set of IPs. One approach is to adapt traditional sequencing methods to detect non-traditional nucleic acids (Figure 2). Recently, engineered polymerases have been de-veloped that can transcribe DNA to a variety of syn-thetic nucleobase polymers, collectively termed xeno-nucleic acids (XNAs), and from XNAs back to DNA [24]. Thus, through transcription of non-traditional IPs to DNA, these IPs can be sequenced.

SETG: The Search for Extraterrestrial Genomes (SETG) instrument, under development, is intended to support in-situ metagenomic or targeted sequencing of RNA or DNA. However, it could be adapted to se-quence other nucleic acid polymers simply by carrying DNA polymerases able to read XNAs. Our current baseline sequencing technology is semiconductor se-quencing [25], in which a small non-optical chip can yield millions of sequences concurrently. By using a metagenomic approach, detection is not limited by our current sequence knowledge. However, we would still rely on converting any RNA or XNAs to DNA before sequencing. A second approach is to utilize a sequenc-ing technology that is not specific to RNA or DNA, such as the nascent technology of nanopores.

Figure 2. Prospects for in-situ sequencing of nucleic acids and other informational polymers using semi-conductor and nanopore sequencing.

Nanopore sequencing: Proposed nanopore devices

can be broadly classified into ionic blockade or trans-conductance devices, named for how one detects pas-sage of an analyte through the nanopore (Figure 3).

In the first device category, ionic blockade, a na-nopore across a highly electrically resistant membrane allows ions to flow from one side of the membrane to the other when a voltage is applied across the mem-brane. When a molecule such as DNA partially blocks the nanopore channel, the ionic current changes, in the ideal, to a specific current that reflects the molecular basis of each monomer within the polymer chain.

Figure 3. Nanopore sequencing overview. A) Biologi-cal nanopore based on ionic blockade. B) Graphene nanogap device based on transconductance.

The channel must be narrow enough that the pres-

ence of a monomer restricts the flow of ions, and also short enough so that the contribution of other mono-mers to the ionic current is limited. In addition, trans-location must be slowed to a speed consistent with measurement of the ionic current, and fluctuations in polymer movement restricted to, in the ideal, unidirec-tional movement without skipping of monomers (sup-pressing Brownian motion).

The second device category, transconductance, re-lys on how the presence of the analyte affects the con-ductance, or equivalently, electrical resistance, across the pore: Simulations show that different DNA bases differentially change the ability of electrons to tunnel across a pore made in a graphene monolayer [26].

Although graphene nanopores could potentially be utilized in either a ionic blockade or transconductance mode, they are the principal theoretical basis to build transconductance devices, in that they offer low noise and a molecularly thin gap that should help to achieve single-nucleotide resolution. As such they are typically called graphene nanogap devices (Figure 3B).

Limitations and Benefits: A question is whether measurements can be uniquely and accurately mapped to individual DNA bases, although Oxford Nanopore has reported error rates of 4% with read lengths up to tens of kilobases (unpublished as of yet). In this sys-tem, phi-29 polymerase performs strand displacement on a double stranded DNA (dsDNA) molecule, rachet-ing the strand into the nanopore base by base in an attempt to provides controlled movement through the pore. Achieving controlled movement in graphene-based nanopores is still a major challenge.

Furthermore, heavy ion bombardment of graphene monolayers shows they can be damaged, even un-zipped, during grazing collisions [27]. This may im-pose design restrictions for space applications such as the need for many nanopores, or for small monolayer exposure areas. In contrast, reagents and chips required for semiconductor sequencing survive analogs of the space radiation environment consistent with a two-year Mars mission.

RNA

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Semiconductor SequencingBiological Nanopore SequencingGraphene Nanogap Sequencing

Other Informational Polymers

DemonstratedTheoretical

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DNA or any appropriatelysized polymer

graphenemonolayer

transconductance(tunneling) current

A B

1136.pdfInternational Workshop on Instrumentation for Planetary Missions (2012)

Arguably the greatest benefit of nanopore sequenc-ing would be the ability to sequence nucleic acids with minimal sample preparation (beyond extraction of nu-cleic acids) and without amplification. It may also be possible to directly sequence both RNA and DNA. Nanopore sequencers may be able to identify modified bases such as methylated cytosine.

Direct XNA sequencing may also be possible: just as engineered polymerases can read and write XNAs to/from DNA, engineering the phi-29 or another poly-merase to strand displace an XNA strand could enable controlled movement of XNAs through the pore. How-ever, it has yet to be demonstrated theoretically or ex-perimentally whether the various XNA monomers would yield unique ionic or transconductance signa-tures.

Finally, even if IPs to be detected are not nucleic acids, as long as they have about the same size and can pass through the pore, inducing current changes that vary with sequence, the informational entropy of raw current traces could demonstrate whether extant IPs exist.

Conclusions: Semiconductor sequencing currently provides a viable, reliable approach to sequence RNA, DNA, and through conversion to DNA, XNA IPs to search for life beyond Earth. If the significant chal-lenges associated with nanopore sequencing can be overcome, this method may provide a way to sequence a host of nucleic acid polymers, and with non-biological approaches, possibly a more general set of IPs. These approaches could be applied to the search for life in environments on Mars in the near-term, En-celadeus in the mid-term, and on Europa in the far-future (Figure 4), reflecting the current state of tech-nology and the relative costs and complexity of these missions. Finally, beyond helping to quantify the ex-tent and diversity of life as we know it, these technolo-gies may also facilitate a search for a shadow bio-sphere here on Earth.

Figure 4. Prospects for sequencing in-situ during fu-ture space missions: where, when, and how?

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"Low" RadiationRNA/DNA/Other IPs

Near-surface DrillSoil/Ice/Brine sample

Near-term mission

ModerateRadiationExtreme Radiation

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or Flyby

Mars

Europa

Mid-termmissionFar future mission

Enceladus

Challenging sampling task

Liquid Sample

SemiconductorSequencing Chip

NanoporeSequencer

Image credits: NASA, Life Technologies, Oxford Nanopore

1136.pdfInternational Workshop on Instrumentation for Planetary Missions (2012)