International Workshop on Instrumentation for Planetary Missions (2012) 1136.pdf

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 [email protected].

Dust, Introduction: Nucleic acid sequencing provides a Comets, Earth GNA/TNA?? RNA DNA powerful approach to search for life beyond Earth, as Meteors Meteoritic Exchange well as to monitor levels of forward contamination [1, Mars Common ancestor / 2nd genesis / no life 2]. A strong case can be made to look for RNA or Sun Enceladeus 2nd genesis / no life DNA-based life on Mars [3, 4]: any life there could Europa 2nd genesis / no life Complex organics Others 2nd genesis / no life have a common ancestor with life on Earth due to ex- produced around all stars Delivered to Life evolved one or tensive meteoritic transfer between our two planets [5- multiple potentially more times 9] and the potential for transfer of viable microbes. habitable zones Here we argue that in-situ sequencing has a role in Figure 1. Delivery of similar organic material to mul- life detection even beyond the context of meteoritic tiple habitable zones: if life arose beyond Earth, what transfer, such as searching for life in potentially habit- informational polymers would it use? 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 As a result, similar organic material may be delivered polymers (IPs). Semiconductor sequencing and the to multiple habitable zones within a given solar sys- imminent arrival of nanopore sequencing may facili- tem. This may bias the evolution of life towards utili- tate in-situ sequencing of RNA, DNA, and other so- zation of a common set of precursor molecules. Thus, called xeno nucleic acid (XNA) polymers. Semicon- if life arose in multiple habitable zones, it may utilize ductor sequencing is reliable and its reagents and chips similar informational polymers (IPs), even in the ab- survive analogs of space radiation consistent with a sence of meteoritic exchange. two-year Mars mission. In contrast, nanopore sequenc- When might the search for RNA or DNA fail? ing is unproven but offers lower bias, simplicity, and Life on Mars, if it developed, could have evolved to the possibility to directly sequence XNAs and perhaps utilize a different IP; however, any Earth-related life even more general IPs. there might have retained the ancestral IP. If such an- Targeting IPs beyond RNA and DNA may yield cestrally-related life first evolved to utilize RNA, life high sensitivity and specificity without assuming that on Mars might be stuck in the RNA world and offer a any life elsewhere uses precisely the same IPs as life snapshot of Earth’s deep past. Due to limited meteorit- on Earth. Discovery of such IPs and their sequences ic exchange, targeting of RNA or DNA would be more would reveal the extent to which life on Earth and likely to fail in potential habitable environments such elsewhere shares a common ancestry or biochemistry. as the probable liquid water oceans beneath Europa Why search for RNA/DNA? Sequencing RNA or [12], Enceladus [10, 11] and possibly Titan [20]. DNA is a powerful approach to characterize life as we Alternatives to RNA/DNA: Although it is possi- know it, and may be able to detect life beyond Earth ble that life beyond Earth could utilize RNA or DNA, also based on these IPs. Significant meteoritic ex- life elsewhere could utilize a different polymer such as change, such as has occurred between Mars and Earth TNA [21, 22] or GNA [23], which have been proposed [5-9], would increase the probability that any life in as possible precursors. For example, life might evolve potential habitable zones would utilize similar or iden- based on an IP that reflects the availability of endemic tical IPs. Thus, sequencing of RNA or DNA may be or delivered organic material or environmental condi- able to detect life under scenarios of shared ancestry. tions that make a specific polymer more advantageous Why look for nucleic acids beyond Mars? Com- (tolerance to pH, salt, temperature, replication fidelity, plex organics including nucleobases or their precursors and other characteristics). In this case, different origins are found in meteorites and cometary samples [13-17] of life could be associated with different IPs. Another and in interstellar space [18]. A major source of these possibility is that different origins may have utilized a organics may be irradiation-induced organic synthesis similar “ancestral” IP but evolved along alternative in stellar nebulae [19], a process that may be common paths, displacing the “ancestral” IP with new polymers, to most stars (Figure 1). or retaining the “ancestral” IP.

International Workshop on Instrumentation for Planetary Missions (2012) 1136.pdf

A B Beyond RNA and DNA: A potentially more sensitive dsDNA DNA or any approach is to enable sequencing of a more broad set phi-29 pol appropriately sized polymer of IPs. One approach is to adapt traditional sequencing α-hemolysin non-conductive graphene transconductance methods to detect non-traditional nucleic acids (Figure membrane monolayer (tunneling) current 2). Recently, engineered polymerases have been de- Ionic current veloped that can transcribe DNA to a variety of syn- thetic nucleobase polymers, collectively termed xeno- Figure 3. Nanopore sequencing overview. A) Biologi- nucleic acids (XNAs), and from XNAs back to DNA cal nanopore based on ionic blockade. B) Graphene [24]. Thus, through transcription of non-traditional IPs nanogap device based on transconductance. 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 The channel must be narrow enough that the pres- RNA or DNA. However, it could be adapted to se- ence of a monomer restricts the flow of ions, and also quence other nucleic acid polymers simply by carrying short enough so that the contribution of other mono- DNA polymerases able to read XNAs. Our current mers to the ionic current is limited. In addition, trans- baseline sequencing technology is semiconductor se- location must be slowed to a speed consistent with quencing [25], in which a small non-optical chip can measurement of the ionic current, and fluctuations in yield millions of sequences concurrently. By using a polymer movement restricted to, in the ideal, unidirec- metagenomic approach, detection is not limited by our tional movement without skipping of monomers (sup- current sequence knowledge. However, we would still pressing Brownian motion). rely on converting any RNA or XNAs to DNA before The second device category, transconductance, re- sequencing. A second approach is to utilize a sequenc- lys on how the presence of the analyte affects the con- ing technology that is not specific to RNA or DNA, ductance, or equivalently, electrical resistance, across such as the nascent technology of nanopores. the pore: Simulations show that different DNA bases differentially change the ability of electrons to tunnel Demonstrated across a pore made in a graphene monolayer [26]. Theoretical Although graphene nanopores could potentially be DNA Sequence DNA Semiconductor Sequencing Biological Nanopore Sequencing utilized in either a ionic blockade or transconductance cDNA Graphene Nanogap Sequencing mode, they are the principal theoretical basis to build synthesis transconductance devices, in that they offer low noise

RNA and a molecularly thin gap that should help to achieve single-nucleotide resolution. As such they are typically XNA called graphene nanogap devices (Figure 3B). Other Informational Polymers Limitations and Benefits: A question is whether measurements can be uniquely and accurately mapped Figure 2. Prospects for in-situ sequencing of nucleic to individual DNA bases, although Oxford Nanopore acids and other informational polymers using semi- has reported error rates of 4% with read lengths up to conductor and nanopore sequencing. tens of kilobases (unpublished as of yet). In this sys- tem, phi-29 polymerase performs strand displacement Nanopore sequencing: Proposed nanopore devices on a double stranded DNA (dsDNA) molecule, rachet- can be broadly classified into ionic blockade or trans- ing the strand into the nanopore base by base in an conductance devices, named for how one detects pas- attempt to provides controlled movement through the sage of an analyte through the nanopore (Figure 3). pore. Achieving controlled movement in graphene- In the first device category, ionic blockade, a na- based nanopores is still a major challenge. nopore across a highly electrically resistant membrane Furthermore, heavy ion bombardment of graphene allows ions to flow from one side of the membrane to monolayers shows they can be damaged, even un- the other when a voltage is applied across the mem- zipped, during grazing collisions [27]. This may im- brane. When a molecule such as DNA partially blocks pose design restrictions for space applications such as the nanopore channel, the ionic current changes, in the the need for many nanopores, or for small monolayer ideal, to a specific current that reflects the molecular exposure areas. In contrast, reagents and chips required basis of each monomer within the polymer chain. for semiconductor sequencing survive analogs of the space radiation environment consistent with a two-year Mars mission. International Workshop on Instrumentation for Planetary Missions (2012) 1136.pdf

Mars Enceladus Arguably the greatest benefit of nanopore sequenc- Semiconductor ing would be the ability to sequence nucleic acids with "Low" Radiation Sequencing Chip Sample minimal sample preparation (beyond extraction of nu- RNA/DNA/Other IPs from Near-surface Drill Orbit cleic acids) and without amplification. It may also be Soil/Ice/Brine sample or possible to directly sequence both RNA and DNA. Near-term mission Flyby Nanopore sequencers may be able to identify modified bases such as methylated cytosine. Direct XNA sequencing may also be possible: just Europa as engineered polymerases can read and write XNAs Liquid Sample to/from DNA, engineering the phi-29 or another poly- Nanopore merase to strand displace an XNA strand could enable Sequencer Moderate Extreme Radiation Radiation controlled movement of XNAs through the pore. How- Challenging sampling task Mid-term ever, it has yet to be demonstrated theoretically or ex- Far future mission mission perimentally whether the various XNA monomers would yield unique ionic or transconductance signa- Image credits: NASA, Life Technologies, Oxford Nanopore Figure 4. Prospects for sequencing in-situ during fu- tures. ture space missions: where, when, and how? Finally, even if IPs to be detected are not nucleic

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