A Plausible Prebiotic Path to Nucleosides: Ribosides and Related Aldosides Generated from Ribulose, Fructose, and Similar Abiotic Precursors.

The prebiotic origins of ribose, nucleosides, and eventually RNA are enduring questions whose answers are central to the RNA world hypothesis. The abiotic synthesis of sugars was first demonstrated over a century ago, but no known prebiotic reaction produces ribose (an aldose sugar) selectively and in good yield. In contrast, ribulose, and fructose (ketose sugars) and other monosaccharides are formed in high yield by several robust abiotic reactions. It is reported here that ketose sugars - both ketopentoses and ketohexoes - serve as precursors for the formation of ribosides and other aldosides, as demonstrated by glycoside-forming reactions involving barbituric acid, a plausibly prebiotic nucleobase. Moreover, a one-pot reaction of glyceraldehyde and barbituric acid was discovered which under mild conditions, and without special minerals or other catalysts, results in the formation of glycosides. These results reveal that an exclusive or high-yielding generation of free ribose was not required for its incorporation into processes that provided the foundations for life.

Water-Soluble Supramolecular Polymers of Paired and Stacked Heterocycles: Assembly, Structure, Properties, and a Possible Path to Pre-RNA.

The hypothesis that RNA and DNA are products of chemical and biological evolution has motivated our search for alternative nucleic acids that may have come earlier in the emergence of life-polymers that possess a proclivity for covalent and non-covalent self-assembly not exhibited by RNA. Our investigations have revealed a small set of candidate ancestral nucleobases that self-assemble into hexameric rosettes that stack in water to form long, twisted, rigid supramolecular polymers. These structures exhibit properties that provide robust solutions to long-standing problems that have stymied the search for a prebiotic synthesis of nucleic acids. Moreover, their examination by experimental and computational methods provides insight into the chemical and physical principles that govern a particular class of water-soluble one-dimensional supramolecular polymers. In addition to efficient self-assembly, their lengths and polydispersity are modulated by a wide variety of positively charged, planar compounds; their assembly and disassembly are controlled over an exceedingly narrow pH range; they exhibit spontaneous breaking of symmetry; and homochirality emerges through non-covalent cross-linking during hydrogel formation. Some of these candidate ancestral nucleobases spontaneously form glycosidic bonds with ribose and other sugars, and, most significantly, functionalized forms of these heterocycles form supramolecular structures and covalent polymers under plausibly prebiotic conditions. This Perspective recounts a journey of discovery that continues to reveal attractive answers to questions concerning the origins of life and to uncover the principles that control the structure and properties of water-soluble supramolecular polymers.

X-ray Fiber Diffraction and Computational Analyses of Stacked Hexads in Supramolecular Polymers: Insight into Self-Assembly in Water by Prospective Prebiotic Nucleobases.

Aqueous solutions of equimolar mixtures of 2,4,6-triaminopyrimidine (TAP) and carboxylic acid substituted cyanuric acid (CyCo6 or R-4MeCyCo6) monomers self-assemble into gel-forming supramolecular polymers. Macroscopic fibers drawn from these mixtures were analyzed by X-ray diffraction to determine their molecular structures. Computational methods were used to explore the intrinsic intermolecular interactions that contribute to the structure and stability of these assemblies. Both polymers are formed by the stacking of hexameric rosettes, (TAP/CyCo6)3 or (TAP/R-4MeCyCo6)3, respectively, into long, stiff, twisted stacks of essentially planar rosettes. Chiral, left-handed supramolecular polymers with a helical twist angle of -26.7° per hexad are formed when the pure enantiomer R-4MeCyCo6 is used. These hexad stacks pack into bundles with a hexagonal crystalline lattice organization perpendicular to the axis of the macroscopic fiber. Polymers formed from TAP and CyCo6, both of which are achiral, assemble into macroscopic domains that are packed as a centered rectangular lattice. Within these domains, the individual polymers exist as either right-handed or left-handed helical stacks, with twist angles of +15° or -15° per hexad, respectively. The remarkable ability of TAP and cyanuric acid derivatives to self-assemble in water, and the structural features of their supramolecular polymers reported here, provide additional support for the proposal that these heterocycles could have served as recognition units for an early form of nucleic acids, before the emergence of RNA.

Depsipeptide Nucleic Acids: Prebiotic Formation, Oligomerization, and Self-Assembly of a New Proto-Nucleic Acid Candidate.

The mechanism by which informational polymers first formed on the early earth is currently unknown. The RNA world hypothesis implies that RNA oligomers were produced prebiotically, before the emergence of enzymes, but the demonstration of such a process remains challenging. Alternatively, RNA may have been preceded by an earlier ancestral polymer, or proto-RNA, that had a greater propensity for self-assembly than RNA, with the eventual transition to functionally superior RNA being the result of chemical or biological evolution. We report a new class of nucleic acid analog, depsipeptide nucleic acid (DepsiPNA), which displays several properties that are attractive as a candidate for proto-RNA. The monomers of depsipeptide nucleic acids can form under plausibly prebiotic conditions. These monomers oligomerize spontaneously when dried from aqueous solutions to form nucleobase-functionalized depsipeptides. Once formed, these DepsiPNA oligomers are capable of complementary self-assembly and are resistant to hydrolysis in the assembled state. These results suggest that the initial formation of primitive, self-assembling, informational polymers on the early earth may have been relatively facile if the constraints of an RNA-first scenario are relaxed.

Spontaneous Symmetry Breaking in the Formation of Supramolecular Polymers: Implications for the Origin of Biological Homochirality.

Aqueous solutions of the achiral, monomeric, nucleobase mimics (2,4,6-triaminopyrimidine, TAP, and a cyanuric acid derivative, CyCo6) spontaneously assemble into macroscopic homochiral domains of supramolecular polymers. These assemblies exhibit a high degree of chiral amplification. Addition of a small quantity of one handedness of a chiral derivative of CyCo6 generates exclusively homochiral structures. This system exhibits the highest reported degree of chiral amplification for dynamic helical polymers or supramolecular helices. Significantly, homochiral polymers comprised of hexameric rosettes with structural features that resemble nucleic acids are formed from mixtures of cyanuric acid (Cy) and ribonucleotides (l-, d-pTARC) that arise spontaneously from the reaction of TAP with the sugars. These findings support the hypothesis that nucleic acid homochirality was a result of symmetry breaking at the supramolecular polymer level.

Glycosylation of a model proto-RNA nucleobase with non-ribose sugars: implications for the prebiotic synthesis of nucleosides.

The emergence of nucleosides is an important, but poorly understood, element of the origins of life. We show that 2,4,6-triaminopyrimidine (TAP), a possible ancestral nucleobase of RNA, is glycosylated in water by non-ribose sugars in yields comparable to those previously reported for its reaction with ribose. The various sugars surveyed include ketoses and aldoses; tetroses, pentoses, and hexoses and are neutral, anionic, or cationic. Though they vary greatly in structure and properties, the data show that all sugars tested form glycosides with TAP. The structures of the eight TAP glycosides formed with glucose and two of its derivatives, glucose-6-phosphate and N-acetylglucosamine, were found to be ß-pyranosides with the glycosylation site on TAP varying with sugar identity. Our results suggest that prebiotic nucleoside formation would not have been restricted to ribose if ancestral RNA (or proto-RNA) utilized TAP and/or other proto-nucleobases with similar reactivities, and that the ability to form higher-order structures may have influenced proto-RNA monomer selection.

Precise sequence control in linear and cyclic copolymers of 2,5-bis(2-thienyl)pyrrole and aniline by DNA-programmed assembly.

A series of linear and cyclic, sequence controlled, DNA-conjoined copolymers of aniline (ANi) and 2,5-bis(2-thienyl)pyrrole (SNS) were synthesized. In one approach, linear copolymers were prepared from complementary DNA oligomers containing covalently attached SNS and ANi monomers. Hybridization of the oligomers aligns the monomers in the major groove of the DNA. Treatment of the SNS- and ANi-containing duplexes with horseradish peroxidase (HRP) and H2O2 causes rapid and efficient polymerization. In this way, linear copolymers (SNS)4(ANi)6 and (ANi)2(SNS)2(ANi)2(SNS)2(ANi)2 were prepared and analyzed. A second approach to the preparation of linear and cyclic copolymers of ANi and SNS employed a DNA encoded module strategy. In this approach, single-stranded DNA oligomers composed of a central region containing (SNS)6 or (ANi)5 covalently attached monomer blocks and flanking 5'- and 3'-single-strand DNA recognition sequences were combined in buffer solution. Self-assembly of these oligomers by Watson-Crick base pairing of the recognition sequences creates linear or cyclic arrays of SNS and ANi monomer blocks. Treatment of these arrays with HRP/H2O2 causes rapid and efficient polymerization to form copolymers having patterns such as cyclic BBA and linear ABA, where B stands for an (SNS)6 block and A stands for an (ANi)5 block. These DNA-conjoined copolymers were characterized by melting temperature analysis, circular dichroism spectroscopy, native and denaturing polyacrylamide gel electrophoresis, and UV-visible-near-IR optical spectroscopy. The optical spectra of these copolymers are typical of those of conducting polymers and are uniquely dependent on the specific order of monomers in the copolymer.

Oxidative thymine mutation in DNA: water-wire-mediated proton-coupled electron transfer.

One-electron oxidation of A/T-rich DNA leads to mutations at thymine. Experimental investigation of DNA containing methyl-deuterated thymine reveals a large isotope effect establishing that cleavage of this carbon-hydrogen bond is involved in the rate-determining step of the reaction. First-principles quantum calculations reveal that the radical cation (electron hole) generated by DNA oxidation, initially located on adenines, localizes on thymine as the proton is lost from the methyl group, demonstrating the role of proton-coupled electron transfer (PCET) in thymine oxidation. Proton transport by structural diffusion along a segmented "water-wire" culminates in proton solvation in the hydration environment, serving as an entropic reservoir that inhibits reversal of the PCET process. These findings provide insight into mutations in A/T-rich DNA such as replication fork stalling that is implicated in early stage carcinogenesis.

Enhanced nonenzymatic ligation of homopurine miniduplexes: support for greater base stacking in a pre-RNA world.

The ancestors of RNA? There is a long-standing proposal that contemporary nucleic acids might have evolved from RNA-like polymers that utilized only purine-purine base pairs. Here we demonstrate the great advantage that increased nucleobase stacking area provides for nonenzymatic ligation.

Structural stabilization of DNA-templated nanostructures: crosslinking with 2,5-bis(2-thienyl)pyrrole monomers.

Ordered 2,5-bis(2-thienyl)pyrrole (SNS) zipper arrays are formed by hybridization of complementary DNA oligomers each containing covalently bound SNS monomers. Upon oxidation with HRP/H(2)O(2), these SNS arrays are converted to oligomers having specific lengths and conformation. As a consequence of this reaction the two SNS-containing strands are permanently crosslinked.

One-Pot Formation of Pairing Proto-RNA Nucleotides and Their Supramolecular Assemblies.

Most contemporary theories for the chemical origins of life include the prebiotic synthesis of informational polymers, including strong interpretations of the RNA World hypothesis. Existing challenges to the prebiotic emergence of RNA have encouraged exploration of the possibility that RNA was preceded by an ancestral informational polymer, or proto-RNA, that formed more easily on the early Earth. We have proposed that the proto-nucleobases of proto-RNA would have readily formed glycosides with ribose and that these proto-nucleosides would have formed base pairs as monomers in aqueous solution, two properties not exhibited by the extant nucleosides or nucleotides. Here we demonstrate that putative proto-nucleotides of the model proto-nucleobases barbituric acid and melamine can be formed in the same one-pot reaction with ribose-5-phosphate. Additionally, the proto-nucleotides formed in these reactions spontaneously form assemblies that are consistent with the presence of Watson-Crick-like base pairs. Together, these results provide further support for the possibility that heterocycles closely related to the extant bases of RNA facilitated the prebiotic emergence of RNA-like molecules, which were eventually replaced by RNA over the course of chemical and biological evolution.

DNA-programmed modular assembly of cyclic and linear nanoarrays for the synthesis of two-dimensional conducting polymers.

Nanometer-scale arrays of conducting polymers were prepared on scaffolds of self-assembling DNA modules. A series of DNA oligomers was prepared, each containing six 2,5-bis(2-thienyl)pyrrole (SNS) monomer units linked covalently to N4 atoms of alternating cytosines placed between leading and trailing 12-nucleobase recognition sequences. These DNA modules were encoded so the recognition sequences would uniquely associate through Watson-Crick assembly to form closed-cycle or linear arrays of aligned SNS monomers. The melting behavior and electrophoretic migration of these assemblies showed cooperative formation of multicomponent arrays containing two to five DNA modules (i.e., 12-30 SNS monomers). The treatment of these arrays with horseradish peroxidase and H(2)O(2) resulted in oxidative polymerization of the SNS monomers with concomitant ligation of the DNA modules. The resulting cyclic and linear arrays exhibited chemical and optical properties typical of conducting thiophene-like polymers, with a red-end absorption beyond 1250 nm. AFM images of the cyclic array containing 18 SNS units revealed highly regular 10 nm diameter objects.

Oxidatively generated damage to DNA at 5-methylcytosine mispairs.

Oxidatively generated damage to DNA has been implicated as causing mutations that lead to aging and disease. The one-electron oxidation of normal DNA leads to formation of a nucleobase radical cation that hops through the DNA until it is trapped irreversibly, primarily by reaction at guanine. It has been observed that 5-methylcytosine (C(m)) is a mutational "hot-spot". However, C(m) in a Watson-Crick base pair with G is not especially susceptible to oxidatively induced damage. Radical cation hopping is inhibited in duplexes that contain C-A or C-T mispairs, but no reaction is detected at cytosine. In contrast, we find that the one-electron oxidation of DNA that contains C(m)-A or C(m)-T mispairs results primarily in reaction at C(m) even in the presence of GG steps. The reaction at C(m) is attributed to proton coupled electron transfer, which provides a relatively low activation barrier path for reaction at 5-methylcytosine. This enhanced reactivity of C(m) in mispairs may contribute to the formation of mutational hot spots at C(m).

Structural and Thermodynamic Control of Supramolecular Polymers and DNA Assemblies with Cyanuric Acid: Influence of Substituents and Intermolecular Interactions.

Understanding the interactions and thermodynamic parameters that govern the structure and stability of supramolecular polymers is challenging because of their flexible nature and high sensitivity to weak intermolecular interactions. The application of both experimental and computational analyses reveals the role that substituents on cyanuric acid (Cy), and other nitrogen-containing heterocycles, play in the formation of novel helical supramolecular structures. In this report, we focus on how noncovalent interactions, including steric and stacking interactions, modulate the structural and physical properties of these assemblies. In-depth analyses and several examples of critical steric and electrostatic effects provide insight into the relationship between intermolecular interactions of Cy with nucleic acids and the structure and thermodynamic stability of the supramolecular polymers they form.

Oxidation of DNA: damage to nucleobases.

All organisms store the information necessary to maintain life in their DNA. Any process that damages DNA, causing a loss or corruption of that information, jeopardizes the viability of the organism. One-electron oxidation is such a process. In this Account, we address three of the central features of one-electron oxidation of DNA: (i) the migration of the radical cation away from the site of its formation; (ii) the electronic and structural factors that determine the nucleobases at which irreversible reactions most readily occur; (iii) the mechanism of reaction for nucleobase radical cations. The loss of an electron (ionization) from DNA generates an electron "hole" (a radical cation), located most often on its nucleobases, that migrates reversibly through duplex DNA by hopping until it is trapped in an irreversible chemical reaction. The particular sequence of nucleobases in a DNA oligomer determines both the efficiency of hopping and the specific location and nature of the damaging chemical reaction. In aqueous solution, DNA is a polyanion because of the negative charge carried by its phosphate groups. Counterions to the phosphate groups (typically Na(+)) play an important role in facilitating both hopping and the eventual reaction of the radical cation with H(2)O. Irreversible reaction of a radical cation with H(2)O in duplex DNA occurs preferentially at the most reactive site. In normal DNA, comprising the four common DNA nucleobases G, C, A, and T, reaction occurs most commonly at a guanine, resulting in its conversion primarily to 8-oxo-7,8-dihydroguanine (8-OxoG). Both electronic and steric effects control the outcome of this process. If the DNA oligomer does not contain a suitable guanine, then reaction of the radical cation occurs at the thymine of a TT step, primarily by a tandem process. The oxidative damage of DNA is a complex process, influenced by charge transport and reactions that are controlled by a combination of enthalpic, entropic, steric, and compositional factors. These processes occur over a broad distribution of energies, times, and spatial scales. The emergence of a complete picture of DNA oxidation will require additional exploration of the structural, kinetic, and dynamic properties of DNA, but this Account offers insight into key elements of this challenge.

Supramolecular assembly-enabled homochiral polymerization of short (dA)n oligonucleotides.

A goal of supramolecular chemistry is to create covalent polymers of precise composition and stereochemistry from complex mixtures by the reversible assembly of specific monomers prior to covalent bond formation. We illustrate the power of this approach with short oligomers of deoxyadenosine monophosphate ((dA)n3'p), n ≥ 3, which form supramolecular assemblies with cyanuric acid. The addition of a condensing agent to these assemblies results in their selective, non-enzymatic polymerization to form long polymers (e.g., (dA)1003'p). Significantly, mixtures of D- and L-(dA)53'p form homochiral covalent polymers, which demonstrates self-sorting of racemic monomers and covalent bond formation exclusively in homochiral assemblies.

One-electron oxidation of DNA: thymine versus guanine reactivity.

One-electron oxidation of anthraquinone (AQ)-linked DNA oligonucleotides containing A/T base pairs with repeating TT steps results in the distance-dependent reaction of the resulting radical cation and base damage at the TT steps that is revealed by subsequent reaction as strand cleavage. However, the inclusion of a remote guanine or GG step inhibits the reaction at thymine and results in predominant reaction at the guanine bases. For the oligomers examined in this work, the results reveal that the specific sequence of nucleobases determines the distance dependence, location of reaction and the efficiency of radical cation migration. In particular, a sequence of A/T base pairs can behave either as a trap, shuttle or barrier, depending on the context of the entire oligomer. The A/T sequences act as a shuttle when reaction occurs at a remote G or GG step and the same sequence of A/T bases acts as a barrier when there is more than one GG step in the sequence. In contrast, the A/T steps act as a trap in sequences that lack guanines.

Reversible Transformation of a Supramolecular Hydrogel by Redox Switching of Methylene Blue-A Noncovalent Chain Stopper.

The simple and reversible control of the degree of polymerization, and thereby the bulk material properties, of a supramolecular polymer is reported. Noncovalent capping agents (chain stoppers) modulate the length of supramolecular polymers by stacking on the surfaces of the polymer's ends. Methylene blue (MB) is a positively charged, planar polycyclic dye that acts as a chain stopper. It can be reversibly switched between its colored, planar, cationic state and a colorless, nonplanar, neutral state (leucomethylene blue, LMB) by reduction with ascorbic acid and then reoxidized to MB by O2. LMB does not act as a chain stopper. This behavior was utilized to reversibly trigger the gel to sol transformation of supramolecular polymers formed by the self-assembly of hexameric rosettes comprising 2,4,6-triaminopyrimidine and a hexanoic acid-substituted cyanuric acid (CyCo6) in aqueous media. The results of our experiments highlight the ability of this approach to reversibly switch between the gel and solution states of materials formed from supramolecular polymers and thereby control their bulk properties.

Oxidatively damaged nucleobases in duplex DNA oligomers: reaction at thymine-thymine mispairs.

Thymine-thymine mispairs are barriers to long-distance radical cation migration and are high reactivity sites in duplex DNA oligomers. A DNA oligomer was prepared that contains only A/T base pairs, arranged into a regularly repeating series of TT steps, and a covalently linked anthraquinone photosensitizer. Its UV irradiation causes the one-electron oxidation of the DNA introducing a radical cation that reacts predominantly at the TT steps as revealed by subsequent strand cleavage. When a remote GG step is introduced into the DNA oligomer, there is little reaction at any of the TT steps and strand cleavage is detected almost exclusively at the GG step. However, when a TT step contains a thymine-thymine mispair, one electron oxidation of the oligomer results in strand cleavage at the mispair and at TT steps preceding it with little reaction at the remote GG step. Experiments in which a thymine in the mispair is replaced by uracil show that the mispair is both a highly reactive site and a barrier to radical cation hopping. These effects of the thymine-thymine mispairs may be associated with its wobble base pair structure.

DNA-directed synthesis of aniline and 4-aminobiphenyl oligomers: programmed transfer of sequence information to a conjoined polymer nanowire.

A new class of materials was prepared from aniline-containing oligomers that are covalently linked to the nucleobases of duplex DNA. Oligomers composed of repeating aniline (PANI) or 4-aminobiphenyl (PAB) units having the properties of conducting polymers conjoined to the DNA were prepared by the reaction of horseradish peroxidase and H2O2 with DNA having the appropriate monomers aligned within the major groove. These oligomers exhibit the spectral and chemical properties typical of para-linked polyanilines. This method of preparation enables utilization of the unique self-recognizing properties and sequence programmability of DNA to create tailored oligomers. This ability was demonstrated experimentally by preparation of PAB oligomers from alternating benzene and aniline monomers. Conjoined conducting polymers carrying the sequence information of DNA may have applicability as nanowires.