Generation of Long RNA Chains in Water

Abstract

The synthesis of RNA chains from 3,5-cAMP and 3,5-cGMP was observed. The RNA chains formed in water, at moderate temperatures (40 -90°C), in the absence of enzymes or inorganic catalysts. As determined by RNase analyses, the bonds formed were canonical 3,5-phosphodiester bonds. The polymerizations are based on two reactions not previously described: 1) oligomerization of 3, 5-cGMP to ϳ25-nucleotide-long RNA molecules, and of 3,5-cAMP to 4-to 8-nucleotide-long molecules. Oligonucleotide A molecules were further extended by reciprocal terminal ligation to yield RNA molecules up to >120 nucleotides long and 2) chain extension by terminal ligation of newly polymerized products of 3,5-cGMP on preformed oligonucleotides. The enzyme-and template-independent synthesis of long oligomers in water from prebiotically affordable precursors approaches the concept of spontaneous generation of (pre)genetic information.The origin of informational polymers is not understood. The RNA polymerization process has been studied for five decades, the results showing that from preactivated precursors polymers of several tens can be obtained, as reviewed previously (1). These pioneering studies provide the proof-of-principle that RNA precursors can self-assemble yielding linear polymers. However, the prebiotic validity of a process based on complex preactivation procedures is limited (1, 2), and the problem of defining a prebiotically plausible chemical and thermodynamic scenario for the synthesis and accumulation of informational polymers remains open. The core of the problem is the standard state Gibbs free energy change (3, 4) stating that condensation reactions are very inefficient in water. Given that extant polymerizations occur in water, this is a major difficulty, only partially solved by the fact that these processes at present occur inside the active site of enzymes where water activity may be drastically reduced. The other part of the extant solution, fruit of evolution, is the use of biologically highly preactivated triphosphate nucleotides (3). In primordia, RNA molecules had no enzymes to catalyze their chain-wise growth, and highly activated precursors can be considered as prebiotic only with difficulty.We reasoned that for a pre-enzymatic polymerization to occur the solution must have relied on a simple and robust process. Ideally, such a process should have been based on compounds that were reactive yet relatively stable, chemically not too elaborate to allow their efficient production, and not too dissimilar from the products of their polymerization to minimize the chemical cost of the process.It was observed that phosphorylation of nucleosides occurs in formamide simply in the presence of a source of organic or inorganic phosphate at temperatures at which both the reactants and the products are stable (5). Phosphorylation occurs in every possible position of the nucleoside sugar moiety resulting, both for purine and pyrimidine nucleosides, in the production of 2Ј-, 3Ј-, 5Ј-, 2Ј,3Ј-cyclic, and 3Ј,5Ј-cyclic XMPs 3 (5). Th...

Formamide and the origin of life

“…We observed [31] that 3 ,5 cyclic guanosine mono-phosphate (3 ,5 cGMP) and to some extent also 3 ,5 cAMP, afford short oligomers upon solubilization in water. Upon treatment in various conditions (see below), these oligomers undergo further polymerization.…”

“…Passing to water chemistry, we describe previous studies and recent advancements on the oligomerization of cyclic purine nucleotides, both in the absence and in the presence of complementary template sequences, and on the nonenzymatic joining of oligomers to form long RNA chains [31]. Interestingly, we have recently observed that RNA polymerization may also occur in the presence of formamide (see Section 2.5).…”

“…The efficiency of the polymerization observed with the Na + form was at least one order of magnitude lower than that observed with the H + form, both in terms of the amount of oligomer formed and of its N avg length. The sample was treated as detailed in [31], the procedure consisting in short of alcohol precipitation, terminal labeling by T4 polynucleotide kinase, phenol extractions and denaturing gel electrophoresis.…”

“…A possible solution of general applicability is provided by the potent concentrating mechanism of both short and long nucleic acid chains by thermal currents through microporus compartments [13]. Yields of the reaction are low, typically resulting in 30 µmoles of oligomers starting from 10 mmoles of monomers (as determined by 31 P NMR titration analysis).…”

“…The polymerization mechanism (to be detailed elsewhere) was determined by (i) characterization of the molecular species produced, as determined by MALDI ToF Mass Spectometry; (ii) 31 P NMR analysis of the type of phosphodiester bond formed; (iii) analysis of the polymerization products by specific ribonucleases. The ensemble of these analyses shows that polymerization occurs by a simple state-of-art mechanism consisting in the formation of 3 ,5 phosphodiester bonds among stacked cyclic nucleotides.…”