Brian J. Cafferty

Efficient Self-Assembly in Water of Long Noncovalent Polymers by Nucleobase Analogues

Molecular self-assembly is widely appreciated to result from a delicate balance between several noncovalent interactions and solvation effects. However, current design approaches for achieving self-assembly in water with small, synthetic molecules do not consider all aspects of the hydrophobic effect, in particular the requirement of surface areas greater than 1 nm(2) for an appreciable free energy of hydration. With the concept of a minimum hydrophobic surface area in mind, we designed a system that achieves highly cooperative self-assembly in water. Two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine) are shown to form extremely long supramolecular polymer assemblies that retain water solubility. The complete absence of intermediate assemblies means that the observed equilibrium is between free monomers and supramolecular assemblies. These observations are in excellent agreement with literature values for the free energy of nucleic acid base interactions as well as the calculated free energy penalty for the exposure of hydrophobic structures in water. The results of our study have implications for the design of new self-assembling structures and hydrogel-forming molecules and may provide insights into the origin of the first RNA-like polymers.

Spontaneous Prebiotic Formation of a β-Ribofuranoside That Self-Assembles with a Complementary Heterocycle

The RNA World hypothesis is central to many current theories regarding the origin and early evolution of life. However, the formation of RNA by plausible prebiotic reactions remains problematic. Formidable challenges include glycosidic bond formation between ribose and the canonical nucleobases, as well as the inability of nucleosides to mutually select their pairing partners from a complex mixture of other molecules prior to polymerization. Here we report a one-pot model prebiotic reaction between a pyrimidine nucleobase (2,4,6-triaminopyrimidine, TAP) and ribose, which produces TAP-ribose conjugates in high yield (60-90%). When cyanuric acid (CA), a plausible ancestral nucleobase, is mixed with a crude TAP+ribose reaction mixture, micrometer-length supramolecular, noncovalent assemblies are formed. A major product of the TAP+ribose reaction is a β-ribofuranoside of TAP, which we term TARC. This nucleoside is also shown to efficiently form supramolecular assemblies in water by pairing and stacking with CA. These results provide a proof-of-concept system demonstrating that several challenges associated with the prebiotic emergence of RNA, or pre-RNA polymers, may not be as problematic as widely believed.

Spontaneous formation and base pairing of plausible prebiotic nucleotides in water

The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life.

Abiotic synthesis of RNA in water: a common goal of prebiotic chemistry and bottom-up synthetic biology.

For more than half a century chemists have searched for a plausible prebiotic synthesis of RNA. The initial advances of the 1960s and 1970s were followed by decades of measured progress and a growing pessimism about overcoming remaining challenges. Fortunately, the past few years have provided a number of important advances, including new abiotic routes for the synthesis of nucleobases, nucleosides, and nucleotides. Recent discoveries also provide additional support for the hypothesis that RNA is the product of evolution, being preceded by ancestral genetic polymers, or pre-RNAs, that are synthesized more easily than RNA. In some cases, parallel searches for plausible prebiotic routes to RNA and pre-RNAs have provided more than one experimentally verified synthesis of RNA substructures and possible predecessors. Just as the synthesis of a contemporary biological molecule cannot be understood without knowledge of cellular metabolism, it is likely that an integrated approach that takes into account both plausible prebiotic reactions and plausible prebiotic environments will ultimately provide the most satisfactory and unifying chemical scenarios for the origin of nucleic acids. In this context, recent advances towards the abiotic synthesis of RNA and candidates for pre-RNAs are beginning to suggest that some molecules (e.g., urea) were multi-faceted contributors to the origin of nucleic acids, and the origin of life.

Darwin's Warm Little Pond: A One‐Pot Reaction for Prebiotic Phosphorylation and the Mobilization of Phosphate from Minerals in a Urea‐Based Solvent

The poor reactivity of insoluble phosphates, such as apatite-group minerals, has been a long-appreciated obstacle for proposed models of prebiotic organophosphate formation. This obstacle presents a significant challenge to the nascent development of an RNA world and other models for the origins of life on Earth. Herein, we demonstrate that a scenario based on the formation of a urea/ammonium formate/water (UAFW) eutectic solution leads to an increase in phosphorylation when compared to urea alone for phosphate sources of varying solubility. In addition, under evaporative conditions and in the presence of MgSO4 , the UAFW eutectic mobilizes the phosphate sequestered in water-insoluble hydroxyapatite, giving rise to a marked increase in phosphorylation. These results suggest that the prebiotic concentrations of urea in a geologically plausible evaporitic environment could solve the problem of organic phosphorylation on a prebiotic Earth.

Ultra-sensitive pH control of supramolecular polymers and hydrogels: pKa matching of biomimetic monomers

Achieving self-assembly in water with small molecules that disassemble in response to minimal changes in environmental conditions is important for the development of many promising materials. We report the most sensitive possible pH control of a supramolecular polymer assembly – demonstrated with hydrogel-forming monomers that mimic nucleic acid base pairing. Bidirectional pH-responsive supramolecular polymers are formed upon the self-assembly of three monomers with two pKa-matched recognition units (one acidic and one basic). These supramolecular assemblies are most stable when the pH equals the pKa of the monomers, but disassemble in response to small pH changes near neutrality (exhibiting the theoretical limit for pH-dependent stability). At pH 7 a hydrogel forms in solutions that are 0.7% by weight in monomer, however, the hydrogel dissolves at pH 6 and pH 8. Additionally, we show that hydrogel stability is finely tuned by employing monomer mixtures that frustrate formation of insoluble aggregates. These results illustrate the advantages of using pKa-matched recognition elements and polymer heterogeneity in the development of responsive materials. Finally, these same recognition elements have recently been proposed as possible ancestors of the current bases of RNA. In this context, the assemblies described here might also provide clues to how the first genetic polymers were driven between states of base pairing and non-base pairing by periodic changes in environmental pH.

Nonenzymatic Ligation of DNA with a Reversible Step and a Final Linkage that Can Be Used in PCR

Nonenzymatic DNA ligation chemistries containing a reversible step allow thermodynamic control of product formation, but they are not necessarily compatible with polymerase enzymes. We report a ligation system that uses commercially available reagents, includes a reversible step, and results in a linkage that can function as a template for PCR amplification with accurate sequence transfer.

Silicate-Promoted Phosphorylation of Glycerol in Non-Aqueous Solvents: A Prebiotically Plausible Route to Organophosphates

Phosphorylation reactions of glycerol were studied using different inorganic phosphates such as sodium phosphate, trimetaphosphate (a condensed phosphate), and struvite. The reactions were carried out in two non-aqueous solvents: formamide and a eutectic solvent consisting of choline-chloride and glycerol in a ratio of 1:2.5. The glycerol reacted in formamide and in the eutectic solvent with phosphate to yield its phosphorylated derivatives in the presence of silicates such as quartz sand and kaolinite clay. The reactions were carried out by heating glycerol with a phosphate source at 85 °C for one week and were analyzed by 31P-nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The yield of the phosphorylated glycerol was improved by the presence of silicates, and reached 90% in some experiments. Our findings further support the proposal that non-aqueous solvents are advantageous for the prebiotic synthesis of biomolecules, and suggest that silicates may have aided in the formation of organophosphates on the prebiotic earth.

20 Supramolecular polymerization of nucleobase-like monomers in water

Elucidating the physiochemical principles that govern molecular self-assembly is of great importance for understanding biological systems and may provide insight into the emergence of the earliest macromolecules of life, an important challenge facing the RNA World hypothesis. Self-assembly results from a delicate balance between multiple noncovalent interactions and solvent effects, but achieving efficient self-assembly in aqueous solution with synthetic molecules has proven particularly challenging. Here, we demonstrate how two physical properties – monomer solubility and large hydrophobic surfaces of intermediate structures – are key elements to achieving supramolecular polymers in aqueous solution (Cafferty et al., 2013). Applying these two principles, we report the highly cooperative self-assembly of two weakly interacting, low molecular weight monomers [cyanuric acid and a modified triaminopyrimidine] into a water-soluble supramolecular assembly (see scheme below). The observed equilibrium between only two appreciably populated states – free monomers and supramolecular assemblies – is in excellent agreement with the values previously determined for the free energy of hydrogen bonding (Klostermeier & Millar, 2002), π − π stacking (Frier et al., 1985), and the calculated free energy penalty for the solvation of hydrophobic structures in water (Chandler, 2005). The similarity of the molecules used in this study for the nucleobases found in contemporary nucleic acids and the demonstration that these monomers assemble while the natural nucleobases do not, suggests that the first informational polymers may have emerged from a similar self-assembly process, if the nucleobases were different then they are today (Hud et al., 2013).