Non-enzymatic, Template-directed Polynucleotide Synthesis
(Dr. Anastassia Kanavarioti, Principal Investigator)
In 1952 Watson and Crick made one of the most enlightened discoveries of our times, i.e. the double helical structure of DNA. More recently, compelling evidence from the frontiers of Molecular Biology suggests that certain RNA molecules, so called ribozymes, have catalytic properties comparable to the ones of enzymes. Considering the fact that RNA can, in principle, be replicated just like DNA, the dual function of RNA as a catalyst and genetic material has led to the proposition of an RNA world. This RNA world, composed of a population of RNA molecules, some of them acting as catalysts and some others as genes, is theorized to have been the precursor of today's DNA/protein world. Although appealing, the proposition of an RNA world has not found wide acceptance among chemists for two reasons: First, because a plausible synthesis of the nucleotide building blocks under prebiotic conditions has yet to be demonstrated and second, because the conditions under which the four natural nucleotides could polymerize to form a population of long RNA molecules are still under investigation.
The issue of non-enzymatic polynucleotide synthesis is the one addressed in this work. There are a number of unsolved problems that are not trivial from an experimenter's point of view. They include a variety of activating substituents or condensing agents which would induce polymerization and potential catalysts ranging from metal ions to clays and from peptides to oligonucleotides, even lipid bilayers. If this complexity were not enough, the nucleotides alone exhibit three nucleophilic sites and therefore are prone to the formation of a pyrophosphate as well as a 2',5'-internucleotide linkage, in addition to the naturally abundant 3',5'-internucleotide linkage.
A mechanism that holds promise in facilitating nucleic acid polymerization in the absence of enzymes is template-directed (TD) synthesis. TD synthesis exploits the presence of a preformed polynucleotide that could direct and catalyze the synthesis of its complementary from mono- or oligonucleotide building blocks. Synthesis of the complementary strand would concurrently accomplish information transfer. Progress in this area has been made, but the incorporation of uridine mononucleotides still remains elusive. Incidentally, our work has recently been highlighted in the UC Santa Cruz Review.
The objectives of the current and future work involve finding (non-enzymatic) conditions that favor (i) oligomerization of mixtures of *pN to form short oligonucleotides, such as (pN)
2 to (pN)6 and (ii) TD elongation of these products with mono- or di-nucleotides. Current work includes the exploitation of nucleotide derivatives that are chemically activated by an imidazole derivative, *pN, and are thus more reactive than the triphosphates, i.e. the natural nucleotide derivatives. The polymerization of *pN is been tested in reactions run in isolation or in mixtures at relatively high concentrations of nucleotide in water and in the presence of metal ions that facilitate the polymerization. Most of the analytical work is done with high performance liquid chromatography (HPLC) using methods that we have pioneered. Identification of products is greatly facilitated by the use of a departmental instrument that combines a state of the art HPLC with a quadrupole mass spectrometer (LC/MS). Future plans include NMR experiments and computer modeling to unravel preferred conformations in the self-association, stacking and hydrogen-bonding between nucleotides.
In the context of TD elongation we have made substantial progress in elucidating the mechanism of TD oligoguanylate, (pG)
n, synthesis on a polycytidylate template, making it the first TD reaction for which a detailed mechanism has been postulated (see, e.g. refs 20 and 29). Kinetic measurements reveal that elongation of a preformed oliguanylate by addition of one mononucleotide, *pG, is facilitated by complexation of at least two *pG molecules downstream (see mechanism). Future work will be aimed in studying other TD reactions and finding conditions that facilitate TD polynucleotide synthesis and incorporation of uridine derivatives.The practical outcome of such investigations will be to find conditions under which polynucleotide synthesis would be facilitated in the absence of protecting side-groups and in environmentally more appealing solvents such as water in place of organic solvents. In addition, the process of going from nucleotide building blocks to nucleic acid polymers in a non-biotic environment is an example of increasing organization and complexity and decreasing entropy, defined here as chemical evolution. Examples of such chemical evolution are hard to come by, but processes such as these must have led to the origin of the protocell and therefore are interesting in the context of the origin of life on Earth and on other planetary bodies.