Demonstration of the abiotic polymerization of ribonucleotides under conditions consistent with conditions that may have existed on the prebiotic Earth is an important goal in "RNA world" research. Recent reports of abiotic RNA polymerization with and without catalysis rely on techniques such as HPLC, gel electrophoresis, and MALDI-TOF MS to analyze the reaction products. It is essential to understand the limitations of these techniques in order to accurately interpret the results of these analyses. In particular, techniques that rely on mass for peak identification may not be able to distinguish between a single, linear RNA oligomer and stable aggregates of smaller linear and/or cyclic RNA molecules. In the case of MALDI-TOF MS, additional complications may arise from formation of salt adducts and MALDI matrix complexes. This is especially true for abiotic RNA polymerization reactions because the concentration of longer RNA chains can be quite low and RNA, as a polyelectrolyte, is highly susceptible to adduct formation and aggregation. Here we focus on MALDI-TOF MS analysis of abiotic polymerization products of imidazole-activated AMP in the presence and absence of montmorillonite clay as a catalyst. A low molecular weight oligonucleotide standard designed for use in MALDI-TOF MS and a 3'-5' polyadenosine monophosphate reference standard were also run for comparison and calibration. Clay-catalyzed reaction products of activated GMP and UMP were also examined. The results illustrate the ambiguities associated with assignment of m/z values in MALDI mass spectra and the need for accurate calibration of mass spectra and careful sample preparation to minimize the formation of adducts and other complications arising from the MALDI process.

Investigations of plausible prebiotic chemistry on early Earth must consider not only chemical reactions to form more complex products such as proto-biopolymers but also reversible, molecular self-assembly that would influence the availability, organization, and sequestration of reactant molecules. The self-assembly of guanosine compounds into higher-order structures and lyotropic liquid crystalline ''gel'' phases through formation of hydrogen-bonded guanine tetrads (G-tetrads) is one such consideration that is particularly relevant to an RNAworld scenario. G-tetrad-based gelation has been well studied for individual guanosine compounds and was recently observed in mixtures of guanosine with 5¢-guanosine monophosphate (GMP) as well. The present work investigates the self-assembly of GMP in the presence of the other RNA nucleotides. Effects of the total concentration and relative proportion of the nucleotides in the mixtures, the form (disodium salt vs. free acid) of the nucleotides, temperature, pH, and salt concentration were determined by visual observations and circular dichroism (CD) spectroscopy. The results show that formation of cholesteric G-tetrad phases is influenced by interactions with other nucleotides, likely through association (e.g., intercalation) of the nucleotides with the Gtetrad structures. These interactions affect the structure and stability of the G-tetrad gel phase, as well as the formation of alternate self-assembled GMP structures such as a continuous, hydrogen-bonded GMP helix or dimers and aggregates of GMP. These interactions and multiple equilibria are influenced by the presence of cations, especially in the presence of K + . This work could have important implications for the emergence of an RNA or proto-RNA world, which would require mixtures of nucleotides at sufficiently high, local concentrations for abiotic polymerization to occur.

Metal is the standard desorption platform for MALDI-MS but other surfaces have been shown to offer advantages for particular types of analytes or applications. One such substrate is fused silica, which has been employed for matrix-free detection of low mass analytes and for affinity MALDI-MS in which binding ligands are immobilized at the fused silica surface. The present work reports improved MALDI-MS detection of RNA oligonucleotides, including polyA, polyU, and polyA/U, at the high end of the mass range when unmodified fused silica is used instead of stainless steel as the MALDI target. The RNA oligonucleotides were abiotically synthesized from activated monomers on catalytic clay surfaces. Further investigation found enhanced signals as well for other anionic biopolymers, including DNA oligonucleotides and heparin. Enhancement also was observed for dextran, which is neutral, indicating that the effect is not restricted to anionic biopolymers. Among more general analytical applications, the results are particularly relevant to rapid screening of abiotic RNA polymerization toward elucidating pathways to life on Earth.

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