Research on the origin of life is highly heterogeneous. After a peculiar historical development, it still includes strongly opposed views which potentially hinder progress. In the 1st Interdisciplinary Origin of Life Meeting, early-career researchers gathered to explore the commonalities between theories and approaches, critical divergence points, and expectations for the future. We find that even though classical approaches and theories—e.g., bottom-up and top-down, RNA world vs. metabolism-first—have been prevalent in origin of life research, they are ceasing to be mutually exclusive and they can and should feed integrating approaches. Here we focus on pressing questions and recent developments that bridge the classical disciplines and approaches, and highlight expectations for future endeavours in origin of life research.

To experimentally test hypotheses about the emergence of living systems from abiotic chemistry, researchers need to be able to run intelligent, automated, and long-term experiments to explore chemical space. Here we report a robotic prebiotic chemist equipped with an automatic sensor system designed for long-term chemical experiments exploring unconstrained multicomponent reactions, which can run autonomously over long periods. The system collects mass spectrometry data from over 10 experiments, with 60 to 150 algorithmically controlled cycles per experiment, running continuously for over 4 weeks. We show that the robot can discover the production of high complexity molecules from simple precursors, as well as deal with the vast amount of data produced by a recursive and unconstrained experiment. This approach represents what we believe to be a necessary step towards the design of new types of Origin of Life experiments that allow testable hypotheses for the emergence of life from prebiotic chemistry.

The formation of giant polyoxometalate (POM) species are relatively underexplored as their self-assembly process is complex due to the rapid kinetics. Polyoxopalladates (POPds) are a class of POMs based on Pd, the largest of which is the {Pd84}Ac wheel, and the slower kinetics mean the system is more amenable to systematic study. Here, we show it is possible to follow the assembly of two types of Pd-wheel; the {Pd84}Gly the smaller {Pd72}Prop wheel formed using glycolate and propionate ligands respectively. We analyzed the formation of {Pd72}Prop and {Pd84}Gly using mass spec-trometry (SEC-HPLC-MS and preparative desalting followed by MS). This was accompanied by studies that followed the chemical shift differences between the outer/inner ligands and the free ligand in solution for the {Pd84}Ac, {Pd72}Prop, and {Pd84}Gly species using NMR; this showed it was possible to track the formation of the wheels. Our findings confirm that the macrocycles assemble from smaller building blocks which react together to form the larger species over a period of days. These findings open the way for further structural derivatives and exploration of their host-guest chemistry.

Life detection in space exploration is strongly influenced by our understanding of life on Earth. However, focusing solely on "life as we know it" risks overlooking traces of unknown life. Instead of searching for specific molecules associated with terrestrial life, we propose prioritizing the detection of universal functional traits of life.Assembly Theory (AT)1 is an agnostic biosignature framework proposing that life produces complex objects in abundance. AT determines the complexity of an object by calculating the smallest number of unique steps required to construct it, based on graph theory. Additionally, the copy number of an object—specific to the investigated environment—is factored in, reflecting how living systems select to produce objects that enhance information storage, stability, or survival. While the theoretical foundation of AT for life detection is well established, particularly for organic molecules, further work is required to use AT for sample interpretation in space exploration. Previous studies have demonstrated AT calculations using data from spectroscopy techniques (IR and NMR)2 and mass spectrometry (direct infusion ESI-MS and LC-MS)1,3. However, LC-MS is currently unsuitable for space missions due to challenges such as solvent weight and the difficulty of mixing solvent gradients in microgravity.Gas chromatography-mass spectrometry (GC-MS) offers a well-established instrument alternative for space exploration3, addressing the limitations of LC-MS while still providing analyte separation. GC-MS was deployed in the Viking mission in 1976, is currently used by Curiosity's SAM instrument, and will be featured in future missions like MOMA on the Rosalind Franklin Rover and DraMS on Dragonfly. Given its heritage and future applications, an experimentally validated GC-MS agnostic biosignature method is urgently needed.Adapting AT estimations for GC-MS requires careful consideration of several parameters, including column selection, derivatization methods and consideration towards sample matrices. We will present preliminary test results of AT estimations using GC-MS and discuss how operational choices may impact the performance of this biosignature detection method. Developing a robust agnostic biosignature method compatible with instruments already deployed provides new opportunities for advancing life detection and interpreting space mission data. 1. Marshall, S.M., Mathis, C., Carrick, E. et al.Identifying molecules as biosignatures with assembly theory and mass spectrometry. Nat Commun 12, 3033 (2021). DOI: 10.1038/s41467-021-23258-x2. Jirasek, M., Sharma, A., Bame, J. R. et al.Investigating and Quantifying Molecular Complexity Using Assembly Theory and Spectroscopy. ACS Central Science (2024). DOI: 10.1021/acscentsci.4c001203. Weiss, G.M., Asche, S., Graham, H.V. et al. Operational considerations for approximating molecular assembly by Fourier transform mass spectrometry. Astron. Space Sci., 11 (2024). DOI: 10.3389/fspas.2024.14854834. Luoth, C., Mahaffy, P., Trainer, M. al. Planetary Mass Spectrometry for Agnostic Life Detection in the Solar System. Front. Astron. Space Sci., 8 (2021). DOI: 10.3389/fspas.2021.755

Assembly theory (AT) is a proposed framework for an agnostic biosignature detection method based on the idea that life produces complex objects in abundance1. The complexity assignment for an object is based on the number of required steps to make the object and derived from graph theory. The consideration for the copy number of that object in the specific environment is based on the idea that functional systems selectively produce objects that are favorable for the systems stability or survival.While the underlying theory of this life detection method is well established, more experimental work is required to prove the theory’s experimental capabilities.Figure 1: Depiction of the required steps for an agnostic biosignature method for life detection. Development of the idea, laboratory testing and deployment.Experimental data has been reported from spectroscopy instruments (IR and NMR) as well as mass spectrometry (MS) instruments (direct injection ESI-MS and LC-MS)2-3. While MS is a promising technology and could enable biosignature research, LC-MS is a technique currently not developed for space exploration due to limitations rising from using solvent as analyte carrier due to their weight but also due to problems creating gradient mixtures in micro gravity.Gas chromatography (GC)-MS is a solution for this problem as no solvent carrier is required and it is a well-established and tested tool for space exploration4-5. It has been deployed in 1968 in the Viking mission, is currently used on Curiosity’s SAM instrument and will be deployed with MOMA on the Rosalind Franklin Rover, with DraMS on Dragonfly and with MASPEX on Europa Clipper6. Because of the amount of experience collected in GC-MS spaceflight and the fact that a higher number of future missions carry this specific instrument, an experimentally tested GC-MS agnostic biosignature method is needed now. We present preliminary results for AT measurements on a GC-Orbitrap-MS. Orbitrap mass spectrometer are widely used for compound identification as they allow for very precise analysis of intact molecules as well as for fragments after additional collision steps. This specific type of MS is currently under development for future lander missions6.Due to the differences between GC-MS and LC-MS or direct infusion, developing a method for AT calculation required consideration towards several parameters. The noise floor reduction and blank subtraction methods needed to be adjusted for GC-MS. Concentration limits were investigated to understand the AT capabilities of the instrument for the specific analytes. Further the choice of columns (polar/ non-polar) and derivatization methods and their impact on the AT calculation result will be presented. Developing an agnostic biosignature method that can be used on instruments that are already in space collecting data offers new opportunities in biosignature development and interpreting collected data. References:1. Sharma, A., Czégel, D., Lachmann, M. et al. Assembly theory explains and quantifies selection and evolution. Nature 622, 321–328 (2023). https://doi.org/10.1038/s41586-023-06600-92. Jirasek, M., Sharma, A., Bame, J. R. et al.Investigating and Quantifying Molecular Complexity Using Assembly Theory and Spectroscopy. ACS Central Science (2024). https://doi.org/1021/acscentsci.4c001203. Marshall, S.M., Mathis, C., Carrick, E. et al.Identifying molecules as biosignatures with assembly theory and mass spectrometry. Nat Commun 12, 3033 (2021). https://doi.org/10.1038/s41467-021-23258-x4. Mahaffy, P.R., Webster, C.R., Cabane, M. et al. The Sample Analysis at Mars Investigation and Instrument Suite. Space Sci Rev 170, 401–478 (2012). https://doi.org/10.1007/s11214-012-9879-z5. Akapo, S. O., Dimandja, J.-M.D., Kojiro, D. R. et al. Gas chromatography in space. Journal of Chromatography A, 843, 1–2, 147-162 (1999).https://doi.org/10.1016/S0021-9673(98)00947-96. Luoth, C., Mahaffy, P., Trainer, M. al. Planetary Mass Spectrometry for Agnostic Life Detection in the Solar System. Frontiers in Astronomy and Space Sciences, 8 (2021). https://doi.org/10.3389/fspas.2021.755