FfAME:org :: Jan Spacek's Publications

Jan Spacek's Publications

Agnostic Life Finder (ALF) for Large-Scale Screening of Martian Life During In Situ Refueling
Spacek, J. & Benner, S.A.
Astrobiology (2022) 22, 8, DOI:10.1089/ast.2021.0070
<Abstract>

Before the first humans depart for Mars in the next decade, hundreds of tons of martian water-ice must be harvested to produce propellant for the return vehicle, a process known as in situ resource utilization (ISRU). We describe here an instrument, the Agnostic Life Finder (ALF), that is an inexpensive life-detection add-on to ISRU. ALF exploits a well-supported view that informational genetic biopolymers in life in water must have two structural features: (1) Informational biopolymers must carry a repeating charge; they must be polyelectrolytes. (2) Their building blocks must fit into an aperiodic crystal structure; the building blocks must be size-shape regular. ALF exploits the first structural feature to extract polyelectrolytes from ?10 cubic meters of mined martian water by applying a voltage gradient perpendicularly to the water's flow. This gradient diverts polyelectrolytes from the flow toward their respective electrodes (polyanions to the anode, polycations to the cathode), where they are captured in cartridges before they encounter the electrodes. There, they can later be released to analyze their building blocks, for example, by mass spectrometry or nanopore. Upstream, martian cells holding martian informational polyelectrolytes are disrupted by ultrasound. To manage the (unknown) conductivity of the water due to the presence of salts, the mined water is preconditioned by electrodialysis using porous membranes. ALF uses only resources and technology that must already be available for ISRU. Thus, life detection is easily and inexpensively integrated into SpaceX or NASA ISRU missions.

A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern
Nguyen, L. T., Macaluso, N. C., Pizzano, B. L., Cash, M. N., Spacek, J., Karasek, Jain, P. K., et. al.
EBioMedicine, Lancet (2022) 77, 103926. DOI:10.1016/j.ebiom.2022.103926

A simple electroanalysis of polyG RNA in mixtures with 3', 5'-cyclic guanosine monophosphate achieved by selective desorption of the monomers from the electrode surface
Hesko, O., Fojta, M., & Spacek, J.
J. Electroanal. Chem, Elsevier (2021) 901, 115773. DOI: 10.1016/j.jelechem.2021.115773
<Abstract>

Previously it has been shown that cyclic nucleoside monophosphates can spontaneously polymerize to form RNA oligonucleotides under conditions simulating prebiotic conditions on Archean Earth. The most efficient polymerization was documented with 3',5'-cyclic guanosine monophosphate (cGMP). In this work a method for fast detection of short polyG RNAs present in a large overabundance of cGMP, modeling conditions in the non-enzymatic nucleotide polymerization mixtures, is presented. The method is based on electrochemical measurements of guanine (G) oxidation signals yielded by RNA oligomers adsorbed onto the surface of a pyrolytic graphite electrode (PGE). To avoid false positive results arising from the G oxidation signals due to co-adsorbed cGMP, a method for selective removal of the monomers from the electrode surface has been devised. In the first step, both cGMP and RNAs are co-adsorbed onto the PGE surface. In the second step, the cGMP is selectively desorbed using treatments in solutions of different tested surfactants (SDS, Tween 20 or Triton X-100), or by washing in deionized water at elevated temperature. We show that this new approach is suitable for selective analysis of products of polymerization reactions from mixtures of their building blocks.

The Limits to Organic Life in the Solar System: From Cold Titan to Hot Venus
Benner, S. A., & Spacek, J.
LPI Contributions, LPI (2021) 2629, 4003.
<Abstract>

Synthetic biology, physical organic chemistry, and other experimental sciences help assess here on Earth the probability of life in alien environments, and guide our search for it in observational and mission astronomy. These have given us "Agnostic Life Finders" (ALFs) for worlds where environments are similar to Earth's. However, they also guide a search for life in exotic environments. We will present by lab experiments showing organic processes that may support life in acidic Venusian clouds.

Chemical guidance in the search for past and extant life on Mars. Decadal Survey in Planetary Sciences and Astrobiology
Benner, S. A., Biondi, E., Kim, H.-J., Spacek, J.
Bulletin of the AAS, AAS (2020) 53(4), DOI:10.3847/25c2cfeb.266df7e7
<Abstract>

NASA should design missions to Mars to generate "Aha!" jolts for scientists researching the molecular origins of life. Recent advances allow these missions to be informed via privileged chemistry that likely generated RNA prebiotically on Earth, as well as general rules that constrain the structure of genetic molecules of extant life on Mars.

Electrochemical Reduction and Oxidation of Eight Unnatural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode
Spacek, J., Karalkar, N., Fojta, M., Wang, J., Benner, S. A
Electrochimica acta, International Society of Electrochemistry (2020) 362:137210, DOI:10.1016/j.electacta.2020.137210
<Abstract>

Recently we showed the reduction and oxidation of six natural 2'-deoxynucleosides in the presence of the ambient oxygen using the very broad potential window of a pyrolytic graphite electrode (PGE). Using the same procedure, 2'-deoxynucleoside analogs (dNs) that are parts of an artificially expanded genetic information system (AEGIS) were analyzed. Seven of the eight tested AEGIS dNs provided specific signals (voltammetric redox peaks). These signals, described here for the first time, will be used in future work to analyze DNA built from expanded genetic alphabets, helping to further develop AEGIS technology and its applications. Comparison of the electrochemical behavior of unnatural dNs with the previously documented behaviors of natural dNs also provides insights into the mechanisms of their respective redox processes.

Electroanalysis of unnatural base pair content in plasmid DNA generated in a semi-synthetic organism
Spacek, J., & Fojta, M.
Electrochimica acta, International Society of Electrochemistry (2020) 364, 137298. DOI: 10.1016/j.electacta.2020.137298
<Abstract>

While standard Watson-Crick base pairing normally involves hydrogen bonds that join size complementary nitrogenous heterocycles, certain unnatural base pairs (UBP) can support replication, transcription, and translation, including these in semi-synthetic organisms (SSO), using size complementarity alone. In this paper we show that the UBPs can be analyzed electrochemically as components of ribo- or 2'-deoxyribonucleosides, corresponding (2'-deoxy)triphosphates, as well as incorporated into DNA. Here, performance of the electrochemical methods profits from the ability of the unnatural bases 5SICS and TPT3 adsorbed at the surface of a mercury electrode to catalyze hydrogen evolution in acidic media. This allows semi-quantitative detection of single UBP within plasmid DNA generated in a SSO. This electrochemical approach provides new way for direct, fast, and low cost UBP analysis and can supplement or replace currently used methods of UBP detection.

Electrochemical Reduction and Oxidation of Six Natural 2'-Deoxynucleosides at a Pyrolytic Graphite Electrode in the Presence or Absence of Ambient Oxygen
Spacek, J., Fojta, M., & Wang, J.
Electroanalysis, Wiley (2019) 31(10), 2057-2066. DOI: 10.1002/elan.201900417
<Abstract>

Recently we have demonstrated that pyrolytic graphite electrodes (PGE) offer a broad analytically useful potential window, ranging from about -2.0V to +1.6V vs. Ag|AgCl|3M KCl, which enables to use the PGE not only for anodic measurements, but also for direct reduction of nucleobases in DNA oligonucleotides. In this follow-up study, we have focused on the electrochemical behavior of four 2'-deoxynucleosides derived from adenine, guanine, cytosine, thymine, uracil, and 5-methyl cytosine on the PGE. On one hand we have obtained analogous primary redox responses as previously with the oligonucleotides. On the other hand, significant differences were observed, particularly when considering secondary responses involving products of the primary conversions, suggesting involvement of different mechanisms. Further we have found that presence of the ambient oxygen in the electrolyte does not dramatically affect the redox signals of the nucleosides. This finding is in contrast with DNA responses measured at the mercury-based electrodes, where deaeration prior to the measurements was necessary. We demonstrate that all studied nucleosides can be analyzed using a simple ex situ (medium exchange) procedure.

Butylacrylate-nucleobase Conjugates as Targets for Two-step Redox Labeling of DNA with an Osmium Tetroxide Complex
Havranova-VidlakovA, P., Spacek, J., Vítova, L., Hermanova, M., Dadova, J., Raindlova, V., Havran, L., et al.
Electroanalysis, Wiley (2018) 30(2), 371-377. DOI: 10.1002/elan.201700702
<Abstract>

Modification of nucleic acids with osmium tetroxide reagents (Os,L, such as OsO₄,2,2'-bipyridine, Os,bpy) has been applied in redox DNA labeling, in probing DNA structure as well as in studies of DNA interactions with other molecules. In natural DNA, primarily thymine residues form adducts with the Os,bpy in a structure selective manner. In this paper we introduce a new two-step technique of DNA modification with the electroactive Os,bpy, consisting in enzymatic construction of DNA bearing butyl acrylate (BA) moieties attached to uracil at C5 or to 7-deaza adenine at C7, followed by chemical modification of a reactive C=C double bond in the acrylate residue. We demonstrate a facile modification of the BA conjugates in both single- and double-stranded (ds) DNA under conditions when modification within the nucleobase rings in ds DNA is hindered. Various DNA-Os,bpy adducts can easily be analyzed electrochemically and distinguished by different redox potentials. The two-step procedure appears to be applicable in osmium redox labelling of ds DNA.

Label-free detection of canonical DNA bases, uracil and 5-methylcytosine in DNA oligonucleotides using linear sweep voltammetry at a pyrolytic graphite electrode
Spacek, J., Danhel, A., Hason, S., & Fojta, M.
Electrochem. commun., Elsevier (2017) 82, 34-38. DOI: 10.1016/j.elecom.2017.07.013
<Abstract>

An innovative approach to label-free voltammetric analysis of DNA at a pyrolytic graphite electrode (PGE) within a broad range of potentials (from - 2.0 to + 1.6 V) in an acetate buffer (pH 5) is presented. Using specifically designed DNA nonamers, we demonstrate not only anodic oxidation, but for the first time also cathodic reduction of nucleobases at the PGE. In addition, products of irreversible oxidation/reduction of the parent bases are shown to yield analytically useful, base-specific cathodic/anodic signals, making it possible to distinguish between the canonical bases (adenine, cytosine, guanine and thymine), uracil (U) and 5-methylcytosine (mC) in DNA. Furthermore, selective electrochemical "switching off" of the redox signals specific to certain nucleobases is presented as a way to resolve overlapping signals. Similarly, newly reported signals corresponding to electrochemically transformed bases can be “switched on” under specific conditions. This approach can be utilized for fast and facile simultaneous label-free analysis of bases in DNA, including mC and U, and to uncover overlapping signals. This significantly extends the possible applications of PGE in DNA research and (bio)sensor development.