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ABSTRACT: The origin of RNA is one of the most formidable problems facing prebiotic chemists. We consider RNA as a product of evolution, as opposed to the more conventional view of RNA as originally being the product of abiotic processes. We have come to accept that life's informational polymers have changed in chemical structure since their emergence, which presents a quandary similar to the paradox of "My Grandfather's Axe". Here, we discuss reasons why all contemporary components of RNA-the nucleobases, ribose, and phosphate-are not likely the original components of the first informational polymer(s) of life. We also evaluate three distinct models put forth as pathways for how the earliest informational polymers might have assembled. We see the quest to uncover the ancestors of RNA as an exciting scientific journey, one that is already providing additional chemical constraints on the origin of life and one that has the potential to produce self-assembling materials, novel catalysis, and bioactive compounds.
Chemistry & biology 04/2013; 20(4):466-74. · 6.52 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 02/2013; · 9.91 Impact Factor
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Chiaolong Hsiao,
Timothy K Lenz,
Jessica K Peters,
Po-Yu Fang,
Dana M Schneider,
Eric J Anderson,
Thanawadee Preeprem,
Jessica C Bowman,
Eric B O'Neill,
Lively Lie,
Shreyas S Athavale,
J Jared Gossett,
Catherine Trippe,
Jason Murray,
Anton S Petrov,
Roger M Wartell,
Stephen C Harvey, Nicholas V Hud,
Loren Dean Williams
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ABSTRACT: Ancient components of the ribosome, inferred from a consensus of previous work, were constructed in silico, in vitro and in vivo. The resulting model of the ancestral ribosome presented here incorporates ∼20% of the extant 23S rRNA and fragments of five ribosomal proteins. We test hypotheses that ancestral rRNA can: (i) assume canonical 23S rRNA-like secondary structure, (ii) assume canonical tertiary structure and (iii) form native complexes with ribosomal protein fragments. Footprinting experiments support formation of predicted secondary and tertiary structure. Gel shift, spectroscopic and yeast three-hybrid assays show specific interactions between ancestral rRNA and ribosomal protein fragments, independent of other, more recent, components of the ribosome. This robustness suggests that the catalytic core of the ribosome is an ancient construct that has survived billions of years of evolution without major changes in structure. Collectively, the data here support a model in which ancestors of the large and small subunits originated and evolved independently of each other, with autonomous functionalities.
Nucleic Acids Research 01/2013; · 8.03 Impact Factor
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ABSTRACT: Satellite tobacco mosaic virus (STMV) is a T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3' end. Here we use selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3' end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.
PLoS ONE 01/2013; 8(1):e54384. · 4.09 Impact Factor
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Anton S Petrov,
Chad R Bernier,
Chiaolong Hsiao,
C Denise Okafor,
Emmanuel Tannenbaum,
Joshua Stern,
Eric Gaucher,
Dana Schneider, Nicholas V Hud,
Stephen C Harvey,
Loren Dean Williams
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ABSTRACT: Some of the magnesium ions in the ribosome are coordinated by multiple rRNA phosphate groups. These magnesium ions link distal sequences of rRNA, primarily by incorporating phosphate groups into the first coordination shell. Less frequently, magnesium interacts with ribosomal proteins. Ribosomal protein L2 appears to be unique by forming specific magnesium-mediated interactions with rRNA. Using optimized models derived from X-ray structures, we subjected rRNA/magnesium/water/rProtein L2 assemblies to quantum mechanical analysis using the density functional theory and natural energy decomposition analysis. The combined results provide estimates of energies of formation of these assemblies, and allow us to decompose the energies of interaction. The results indicated that RNA immobilizes magnesium by multidentate chelation with phosphate, and that the magnesium ions in turn localize and polarize water molecules, increasing energies and specificities of interaction of these water molecules with L2 protein. Thus, magnesium plays subtle, yet important, roles in ribosomal assembly beyond neutralization of electrostatic repulsion and direct coordination of RNA functional groups.
The Journal of Physical Chemistry B 06/2012; · 3.70 Impact Factor
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ABSTRACT: Structures formed by human telomere sequence (HTS) DNA are of interest due to the implication of telomeres in the aging process and cancer. We present studies of HTS DNA folding in an anhydrous, high viscosity deep eutectic solvent (DES) comprised of choline choride and urea. In this solvent, the HTS DNA forms a G-quadruplex with the parallel-stranded ("propeller") fold, consistent with observations that reduced water activity favors the parallel fold, whereas alternative folds are favored at high water activity. Surprisingly, adoption of the parallel structure by HTS DNA in the DES, after thermal denaturation and quick cooling to room temperature, requires several months, as opposed to less than 2 min in an aqueous solution. This extended folding time in the DES is, in part, due to HTS DNA becoming kinetically trapped in a folded state that is apparently not accessed in lower viscosity solvents. A comparison of times required for the G-quadruplex to convert from its aqueous-preferred folded state to its parallel fold also reveals a dependence on solvent viscosity that is consistent with Kramers rate theory, which predicts that diffusion-controlled transitions will slow proportionally with solvent friction. These results provide an enhanced view of a G-quadruplex folding funnel and highlight the necessity to consider solvent viscosity in studies of G-quadruplex formation in vitro and in vivo. Additionally, the solvents and analyses presented here should prove valuable for understanding the folding of many other nucleic acids and potentially have applications in DNA-based nanotechnology where time-dependent structures are desired.
Journal of the American Chemical Society 06/2012; 134(37):15324-30. · 9.91 Impact Factor
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Current Opinion in Structural Biology 05/2012; 22(3):262-72. · 9.42 Impact Factor
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ABSTRACT: 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.
ChemBioChem 05/2012; 13(8):1121-4. · 3.94 Impact Factor
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Shreyas S Athavale,
J Jared Gossett,
Chiaolong Hsiao,
Jessica C Bowman,
Eric O'Neill,
Eli Hershkovitz,
Thanawadee Preeprem, Nicholas V Hud,
Roger M Wartell,
Stephen C Harvey,
Loren Dean Williams
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ABSTRACT: The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU. Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained within the intact 23S rRNA using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), in the absence and presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.
RNA 02/2012; 18(4):752-8. · 5.09 Impact Factor
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Shreyas S Athavale,
Anton S Petrov,
Chiaolong Hsiao,
Derrick Watkins,
Caitlin D Prickett,
J Jared Gossett,
Lively Lie,
Jessica C Bowman,
Eric O'Neill,
Chad R Bernier, Nicholas V Hud,
Roger M Wartell,
Stephen C Harvey,
Loren Dean Williams
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ABSTRACT: Mg²⁺ shares a distinctive relationship with RNA, playing important and specific roles in the folding and function of essentially all large RNAs. Here we use theory and experiment to evaluate Fe²⁺ in the absence of free oxygen as a replacement for Mg²⁺ in RNA folding and catalysis. We describe both quantum mechanical calculations and experiments that suggest that the roles of Mg²⁺ in RNA folding and function can indeed be served by Fe²⁺. The results of quantum mechanical calculations show that the geometry of coordination of Fe²⁺ by RNA phosphates is similar to that of Mg²⁺. Chemical footprinting experiments suggest that the conformation of the Tetrahymena thermophila Group I intron P4-P6 domain RNA is conserved between complexes with Fe²⁺ or Mg²⁺. The catalytic activities of both the L1 ribozyme ligase, obtained previously by in vitro selection in the presence of Mg²⁺, and the hammerhead ribozyme are enhanced in the presence of Fe²⁺ compared to Mg²⁺. All chemical footprinting and ribozyme assays in the presence of Fe²⁺ were performed under anaerobic conditions. The primary motivation of this work is to understand RNA in plausible early earth conditions. Life originated during the early Archean Eon, characterized by a non-oxidative atmosphere and abundant soluble Fe²⁺. The combined biochemical and paleogeological data are consistent with a role for Fe²⁺ in an RNA World. RNA and Fe²⁺ could, in principle, support an array of RNA structures and catalytic functions more diverse than RNA with Mg²⁺ alone.
PLoS ONE 01/2012; 7(5):e38024. · 4.09 Impact Factor
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ABSTRACT: Increasingly exact measurement of single crystal X-ray diffraction data offers detailed characterization of DNA conformation, hydration and electrostatics. However, instead of providing a more clear and unambiguous image of DNA, highly accurate diffraction data reveal polymorphism of the DNA atomic positions and conformation and hydration. Here we describe an accurate X-ray structure of B-DNA, painstakingly fit to a multistate model that contains multiple competing positions of most of the backbone and of entire base pairs. Two of ten base-pairs of CCAGGCCTGG are in multiple states distinguished primarily by differences in slide. Similarly, all the surrounding ions are seen to fractionally occupy discrete competing and overlapping sites. And finally, the vast majority of water molecules show strong evidence of multiple competing sites. Conventional resolution appears to give a false sense of homogeneity in conformation and interactions of DNA. In addition, conventional resolution yields an average structure that is not accurate, in that it is different from any of the multiple discrete structures observed at high resolution. Because base pair positional heterogeneity has not always been incorporated into model-building, even some high and ultrahigh-resolution structures of DNA do not indicate the full extent of conformational polymorphism.
Nucleic Acids Research 12/2011; 40(8):3714-22. · 8.03 Impact Factor
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ABSTRACT: Many high school laboratory experiments demonstrate concepts related to biological evolution, but few exist that allow students to investigate life's chemical origins. This series of laboratory experiments has been developed to allow students to explore and appreciate the deep connection that exists between prebiotic chemistry, chemical evolution, and contemporary biological systems. In the first experiment of the series, students synthesize adenine, one of the purine nucleobases of DNA and RNA, from plausibly prebiotic precursor molecules. Students compare their product to authentic standards using thin-layer chromatography. The second and third experiments of the series allow students to extract DNA from a familiar organism, the strawberry, and hydrolyze it, releasing adenine, which they can then compare to the previously chemically-synthesized adenine. A fourth, optional experiment is included where the technique of thin-layer chromatography is introduced and chromatographic skills are developed for use in the other three experiments that comprise this series. Concepts relating to organic and analytical chemistry, as well as biochemistry and DNA structure, are incorporated throughout, allowing this series of laboratory experiments to be easily inserted into existing laboratory courses and to reinforce concepts already included in any high school chemistry or biology curriculum.
Journal of chemical education 12/2011; 88(12):1698-1701. · 0.74 Impact Factor
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ChemBioChem 07/2011; 12(14):2155-8. · 3.94 Impact Factor
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Angewandte Chemie International Edition 08/2010; 49(36):6310-4. · 13.45 Impact Factor
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ChemBioChem 06/2010; 11(9):1240-3. · 3.94 Impact Factor
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ABSTRACT: Since the structure of DNA was elucidated more than 50 years ago, Watson-Crick base pairing has been widely speculated to be the likely mode of both information storage and transfer in the earliest genetic polymers. The discovery of catalytic RNA molecules subsequently provided support for the hypothesis that RNA was perhaps even the first polymer of life. However, the de novo synthesis of RNA using only plausible prebiotic chemistry has proven difficult, to say the least. Experimental investigations, made possible by the application of synthetic and physical organic chemistry, have now provided evidence that the nucleobases (A, G, C, and T/U), the trifunctional moiety ([deoxy]ribose), and the linkage chemistry (phosphate esters) of contemporary nucleic acids may be optimally suited for their present roles-a situation that suggests refinement by evolution. Here, we consider studies of variations in these three distinct components of nucleic acids with regard to the question: Is RNA, as is generally acknowledged of DNA, the product of evolution? If so, what chemical and structural features might have been more likely and advantageous for a proto-RNA?
Cold Spring Harbor perspectives in biology 05/2010; 2(12):a002196. · 9.40 Impact Factor
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ABSTRACT: The RNA world hypothesis proposes that nucleic acids were once responsible for both information storage and chemical catalysis, before the advent of coded protein synthesis. However, it is difficult to imagine how nucleic acid polymers first appeared, as the abiotic chemical formation of long nucleic acid polymers from mononucleotides or short oligonucleotides remains elusive, and barriers to achieving this goal are substantial. One specific obstacle to abiotic nucleic acid polymerization is strand cyclization. Chemically activated short oligonucleotides cyclize efficiently, which severely impairs polymer growth. We show that intercalation, which stabilizes and rigidifies nucleic acid duplexes, almost totally eliminates strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length. In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra- and octanucleotides are produced. Intercalator-free polymerization is not observed, even at tetranucleotide concentrations > 10,000-fold greater than those at which intercalators enable polymerization. We also demonstrate that intercalation-mediated polymerization is most favored if the size of the intercalator matches that of the base pair; intercalators that bind to Watson-Crick base pairs promote the polymerization of oligonucleotides that form these base pairs. Additionally, we demonstrate that intercalation-mediated polymerization is possible with an alternative, non-Watson-Crick-paired duplex that selectively binds a complementary intercalator. These results support the hypothesis that intercalators (acting as 'molecular midwives') could have facilitated the polymerization of the first nucleic acids and possibly helped select the first base pairs, even if only trace amounts of suitable oligomers were available.
Proceedings of the National Academy of Sciences 03/2010; 107(12):5288-93. · 9.68 Impact Factor
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ABSTRACT: The problem of beta-nucleoside formation under prebiotic conditions represents one of the most significant challenges to the "RNA world" hypothesis. The possibility exists that alternative bases may have come before the contemporary bases (i.e., A, G, C, and U), including bases that more readily form nucleosides. We previously reported the first successful synthesis of a pyrimidine nucleoside from a free base and a nonactivated sugar in a plausible prebiotic reaction. Here we present a detailed computational study on the reaction at the density functional theory (DFT) level. The catalytic role of a Mg(2+) ion on the reaction mechanism is also investigated. Our calculations demonstrate that a Mg(2+) ion, serving as a Lewis acid, can afford the necessary stabilization to the base and leaving water molecule during glycoside bond formation. The solvent effect is considered by the Onsager solvation model and also by an extended model with the addition of explicit water molecules within the SCRF solvation model. In addition, predictions regarding the formation of nucleosides from other pyrimidine bases are also addressed, providing valuable insights into what chemical features of the bases facilitate glycoside formation in drying-heating reactions.
Journal of the American Chemical Society 11/2009; 131(44):16088-95. · 9.91 Impact Factor
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ABSTRACT: Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson-Crick DNA. Hud's laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine-adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson-Crick Hoogsteen (transWH) or Watson-Crick Watson-Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA-->7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A.A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA-->7 mutations, these results are consistent with the N1-N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.
Nucleic Acids Research 10/2009; 37(22):7715-27. · 8.03 Impact Factor
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ABSTRACT: As a means to explore the influence of the nucleic acid backbone on the intercalative binding of ligands to DNA and RNA, we have determined the solution structure of a proflavine-bound 2',5'-linked octamer duplex with the sequence GCCGCGGC. This structure represents the first NMR structure of an intercalated RNA duplex, of either backbone structural isomer. By comparison with X-ray crystal structures, we have identified similarities and differences between intercalated 3',5' and 2',5'-linked RNA duplexes. First, the two forms of RNA have different sugar pucker geometries at the intercalated nucleotide steps, yet have the same interphosphate distances. Second, as in intercalated 3',5' RNA, the phosphate backbone angle zeta at the 2',5' RNA intercalation site prefers to be in the trans conformation, whereas unintercalated 2',5' and 3',5' RNA prefer the -gauche conformation. These observations provide new insights regarding the transitions required for intercalation of a phosphodiester-ribose backbone and suggest a possible contribution of the backbone to the origin of the nearest-neighbor exclusion principle. Thermodynamic studies presented for intercalation of both structural RNA isomers also reveal a surprising sensitivity of intercalator binding enthalpy and entropy to the details of RNA backbone structure.
Journal of the American Chemical Society 04/2009; 131(16):5831-8. · 9.91 Impact Factor
