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Project Progress

Our research in the past year has focused on the following aspects of prebiotic earth and abiotic RNA synthesis:

Potential pitfalls in MALDI-MS analysis of abiotically produced RNA polymerization products
The MALDI-MS technique is much better characterized and optimized for analysis of proteins than for analysis of oligonucleotides. Interference from the MALDI matrix is not limited to background signal but may also promote formation of clusters of ribonucleotides. These clusters could be mistaken for actual polymerization products. Separate analysis by HPLC can confirm interpretation of MALDI-MS results but this is at the expense of the potentially superior detectability of MALDI-MS that could detect longer oligomers. We are performing systematic studies of MALDI-MS analysis of RNA polymerization products along with oligomer standards in order to improve the reliability of this approach for studying abiotic RNA synthesis.

Why G?
Regardless of whether RNA is the original molecule of life or evolved early on from less optimal precursors, it is extremely likely that it played a key role in the earliest stages of life on earth. It is important to consider the ultimate selection of each of the components of RNA ribonucleotides: why these particular nucleobases? Why ribose? Why phosphate? In particular, we are fascinated and puzzled by the inclusion of guanosine monophosphate (GMP) among the four ribonucleotides. This is because guanosine, GMP and other guanosine derivatives exhibit a unique property of reversible self-aggregation to form guanosine tetrads that can further associate through pi-pi stacking to form chiral, columnar aggregates and, at higher concentrations, lyotropic liquid crystalline phases. This alternate pathway for GMP competes with its incorporation into oligonucleotides, which is why it is difficult to synthesize highly G-rich RNA or DNA with good efficiency.

Given the competing pathway for GMP, we can ask if G is one of the four ribonucleotides in RNA in spite of, or because of, its unique properties. Our hypothesis is that the answer is “because of”. For one thing, G obviously has been quite successful in both RNA and DNA. But more intriguing is the possibility that G served a regulatory function that guided RNA sequences in a particular direction among the astronomically large combinatorial space of possibilities. This, combined with the ability of some G-rich sequences to form highly stable, intramolecular G-quadruplex structures that have been observed in aptamers, telomeres, and numerous G-rich sequences from genomic DNA, is the basis for our hypothesis. There are many interesting factors to consider in this work, including the tendency of guanosine compounds to form highly viscous gels at high monomer concentrations, especially in the presence of cations, particularly K⁺ and Na⁺.

We are studying the gelation of GMP in the presence of the XMP (X=A, U, C). We are also testing the ability of GMP alone and GMP/XMP mixtures to disperse catalytic clays. Differences in miscibility of the different nucleotides in GMP are an important feature of a scenario in which GMP would exert selective pressure on RNA sequence. The compatibility of catalytic minerals with these combined nucleotide phases is essential to the catalytic role of the minerals.

Effects of environmental conditions and components on abiotic RNA polymerization
Here we are interested in the effects of compounds that may have been present in the prebiotic environment during abiotic RNA synthesis. Our initial focus is on amino acids. We titrated montmorillonite clays with either alanine or tryptophan to determine any effects on RNA polymerization. We are in the process of analyzing the results using HPLC and MALDI MS.