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All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

Relative reactivity of ribosyl 2'-OH vs. 3'-OH in concentrated aqueous solutions of phosphoimidazolide activated nucleotides

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN2' pN and pN3' pN) and the pyrophosphate-linked (N5' ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2'-OH vs. 3'-OH was determined from the ratio of the yields of pN2' pN vs. pN3' pN. Experiments were performed at 23 degrees C in the range 7.2 < or = pH < or = 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg2+ or Mn2+) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN2' pN: pN3' pN = 2'-5': 3'-5' was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2'-OH is about 2 to 3 times more reactive than the 3'-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN2' pN: pN3' pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN2' pN: pN3' pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5' = 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3'-OH of one molecule to the reactive P-N bond of an other molecule. The implication of these conclusions for oligomerization/ligation reactions is discussed.

NASA Discipline Exobiology↗
NASA NTRS1998

Preference for internucleotide linkages as a function of the number of constituents in a mixture

Phosphoimidazolide-activated ribomononucleotides (*pN; see Scheme I) are useful substrates for the nonenzymatic synthesis of oligonucleotides. In the presence of metal ions dilute neutral aqueous solutions of *pN (0.01 M) typically yield only small amounts of dimers and traces of oligomers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate (5'NMP). An earlier investigation of *pN reactions in highly concentrated aqueous solutions (up to 1.4 M) showed, as expected, that the percentage yield of the condensation products increases and the yield of the hydrolysis product correspondingly decreases with *pN concentration (Kanavarioti 1997). Here we report product distributions in reactions with one, two, or three reactive components at the same total nucleotide concentration. *pN used as substrates were the nucleoside 5'-phosphate 2-methylimidazolides, 2-MeImpN, with N = cytidine (C), uridine (U), or guanosine (G). Reactions were conducted as self-condensations, i. e., one nucleotide only, with two components in the three binary U,C, U,G, and C,G mixtures, and with three components in the ternary U,C, G mixture. The products are 5'NMP, 5',5'-pyrophosphate-, 2',5'-, 3', 5'-linked dimers, cyclic dimers, and a small percentage of longer oligomers. The surprising finding was that, under identical conditions, including the same total monomer concentration, the product distribution differs substantially from one reaction to another, most likely due to changing intermolecular interactions depending on the constituents. Even more unexpected was the observed trend according to which reactions of the U,C,G mixture produce the highest yield of internucleotide-linked dimers, whereas the self-condensations produce the least and the reactions with the binary mixtures produce yields that fall in between. What is remarkable is that the approximately two-fold increase in the percentage yield of internucleotide-linked dimers is not due to a concentration effect or a catalyst, but to the increased complexity of the system from a single to two and three components. These observations, perhaps, provide an example of how increased complexity in relatively simple chemical systems leads to organization of the material and consequently to chemical evolution. A possible link between prebiotic chemistry and the postulated RNA world is discussed.

NASA Discipline Exobiology↗
NASA NTRS1980

A theory for the origin of a self-replicating chemical system. I - Natural selection of the autogen from short, random oligomers

A general theory is presented for the origin of a self-replicating chemical system, termed an autogen, which is capable of both crude replication and translation (protein synthesis). The theory requires the availability of free energy and monomers to the system, a significant background low-yield synthesis of kinetically stable oligopeptides and oligonucleotides, the localization of the oligomers, crude oligonucleotide selectivity of amino acids during oligopeptide synthesis, crude oligonucleotide replication, and two short peptide families which catalyze replication and translation, to produce a localized group of at least one copy each of two protogenes and two protoenzymes. The model posits a process of random oligomerization, followed by the random nucleation of functional components and the rapid autocatalytic growth of the functioning autogen to macroscopic amounts, to account for the origin of the first self-replicating system. Such a process contains steps of such high probability and short time periods that it is suggested that the emergence of an autogen in a laboratory experiment of reasonable time scale may be possible.

White, D. H.↗
NASA NTRS1991

Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

Wu, Taifeng↗
NASA NTRS1999

Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization

Hexitol nucleic acids (HNAs) are DNA analogues that contain the standard nucleoside bases attached to a phosphorylated 1,5-anhydrohexitol backbone. We find that HNAs support efficient information transfer in nonensymatic template-directed reactions. HNA heterosequences appeared to be superior to the corresponding DNA heterosequences in facilitating synthesis of complementary oligonucleotides from nucleoside-5'-phosphoro-2-methyl imidazolides.

Non-NASA Center↗
NASA NTRS1986

Molecular replication

The object of our research program is to understand how polynucleotide replication originated on the primitive Earth. This is a central issue in studies of the origins of life, since a process similar to modern DNA and RNA synthesis is likely to have formed the basis for the most primitive system of genetic information transfer. The major conclusion of studies so far is that a preformed polynucleotide template under many different experimental conditions will facilitate the synthesis of a new oligonucleotide with a sequence complementary to that of the template. It has been shown, for example, that poly(C) facilitates the synthesis of long oligo(G)s and that the short template CCGCC facilities the synthesis of its complement GGCGG. Very recently we have shown that template-directed synthesis is not limited to the standard oligonucleotide substrates. Nucleic acid-like molecules with a pyrophosphate group replacing the phosphate of the standard nucleic acid backbone are readily synthesized from deoxynucleotide 3'-5'-diphosphates on appropriate templates.

Orgel, L. E.↗
NASA NTRS1986

Stereoselective aminoacylation of RNA

Prebiotic chemistry is faced with a major problem: how could a controlled and selective reaction occur, when there is present in the same solution a large number of alternative possible coreactants? This problem is solved in the modern cell by the presence of enzymes, which are not only highly efficient and controllable catalysts, but which also can impose on their substrates a precise structural requirement. However, enzymes are the result of billions of years of evolution, and we cannot invoke them as prebiotic catalysts. One approach to solving this problem in the prebiotic context is to make use of template-directed reactions. These reactions increase the number of structural requirements that must be simultaneously present in a molecule for it to be able to react, and thereby increase the selectivity of the reaction. They also can give a large increase in the rate of a reaction, if the template constrains two potential coreactants to lie close together. A third benefit is that information that is present in the template molecule can be passed on to the product molecules. If the earliest organisms were based on proteins and nucleic acids, then the investigation of peptide synthesis on an oligonucleotide template is highly relevant to the study of the origin of life.

Usher, D. A.↗
DOE OSTI2023

Synthesis and Structural Characterization of 2′‐Deoxy‐2′‐Methoxy‐ L ‐Cytidine Nucleic Acids

Abstract We report here for the first time the synthesis of a novel 2′‐methoxy‐modified L‐cytidine analog and its phosphoramidite, as well as its incorporation into oligonucleotides via solid‐phase synthesis. Our measure the thermostability of the modified L‐nucleic acids by UV‐melting studies, and find out the 2′‐methoxy‐derivatization enhances the thermostability of the L‐duplexes. Consistent with the thermostability result, our X‐ray crystal structure study reveals that the 2′‐methoxy‐L‐deoxycytidine forms the Watson‐Crick base pair with L‐guanosine virtually as the mirror‐image fashion of native pair. This novel chemically modified L‐cytidine provides a useful biochemical and structural strategy to create mirror‐image nucleic acid molecules with expanded functionalities.

2′-Methoxy↗
NASA NTRS1976

Abiotic origin of biopolymers

A variety of methods have been investigated in different laboratories for the polymerization of amino acids and nucleotides under abiotic conditions. They include (1) thermal polymerization; (2) direct polymerization of certain amino acid nitriles, amides, or esters; (3) polymerization using polyphosphate esters; (4) polymerization under aqueous or drying conditions at moderate temperatures using a variety of simple catalysts or condensing agents like cyanamide, dicyandiamide, or imidazole; and (5) polymerization under similar mild conditions but employing activated monomers or abiotically synthesized high-energy compounds such as adenosine 5'-triphosphate (ATP). The role and significance of these methods for the synthesis of oligopeptides and oligonucleotides under possible primitive-earth conditions is evaluated. It is concluded that the more recent approach involving chemical processes similar to those used by contemporary living organisms appears to offer a reasonable solution to the prebiotic synthesis of these biopolymers.

Oro, J.↗
NASA NTRS2003

5' modification of duplex DNA with a ruthenium electron donor-acceptor pair using solid-phase DNA synthesis

Incorporation of metalated nucleosides into DNA through covalent modification is crucial to measurement of thermal electron-transfer rates and the dependence of these rates with structure, distance, and position. Here, we report the first synthesis of an electron donor-acceptor pair of 5' metallonucleosides and their subsequent incorporation into oligonucleotides using solid-phase DNA synthesis techniques. Large-scale syntheses of metal-containing oligonucleotides are achieved using 5' modified phosporamidites containing [Ru(acac)(2)(IMPy)](2+) (acac is acetylacetonato; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (3) and [Ru(bpy)(2)(IMPy)](2+) (bpy is 2,2'-bipyridine; IMPy is 2'-iminomethylpyridyl-2'-deoxyuridine) (4). Duplexes formed with the metal-containing oligonucleotides exhibit thermal stability comparable to the corresponding unmetalated duplexes (T(m) of modified duplex = 49 degrees C vs T(m) of unmodified duplex = 47 degrees C). Electrochemical (3, E(1/2) = -0.04 V vs NHE; 4, E(1/2) = 1.12 V vs NHE), absorption (3, lambda(max) = 568, 369 nm; 4, lambda(max) = 480 nm), and emission (4, lambda(max) = 720 nm, tau = 55 ns, Phi = 1.2 x 10(-)(4)) data for the ruthenium-modified nucleosides and oligonucleotides indicate that incorporation into an oligonucleotide does not perturb the electronic properties of the ruthenium complex or the DNA significantly. In addition, the absence of any change in the emission properties upon metalated duplex formation suggests that the [Ru(bpy)(2)(IMPy)](2+)[Ru(acac)(2)(IMPy)](2+) pair will provide a valuable probe for DNA-mediated electron-transfer studies.

Non-NASA Center↗
DOE OSTI2025

MIST_paper

Code to reproduce results from and implement functionality described in "A Bayesian error model for synthesis and sequencing of oligonucleotides", Marrs, FW, Gratz, D, and Erkkila, TH.

Marrs, Frank↗
NASA NTRS1981

A model for protocellular coordination of nucleic acid and protein syntheses

The proteinoid model for the coordination of protein synthesis with nucleic acid coding within the evolving protocell is discussed. Evidence for the self-ordering of amino acid chains, which would enhance the catalytic activity of a lysine-rich proteinoid, is presented, along with that for the preferential formation of microparticles, particularly proteinoid microparticles, in various solutions. Demonstrations of the catalytic activity of lysine-rich proteinoids in the synthesis of peptide and internucleotide bonds are pointed out. The view of evolution as a two stage sequence in which the geological synthesis of peptides evolved to the protocellular synthesis of peptides and oligonucleotides is discussed, and contrasted with the alternative view, in accord with the central dogma, that nucleic acids arose first then governed the production of proteins and protocells.

Fox, S. W.↗
NASA NTRS1995

Template switching between PNA and RNA oligonucleotides

The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

NASA Discipline Exobiology↗
NASA NTRS1991

Product and rate determinations with chemically activated nucleotides in the presence of various prebiotic materials, including other mono- and polynucleotides

We are investigating the reactions of ImpN's in the presence of a number of prebiotically plausible materials, such as metal ions, phosphate, amines and other nucleotides and hope to learn more about the stability/reactivity of ImpN's in a prebiotic aqueous environment. We find that, in the presence of phosphate, ImpN's form substantial amounts of diphosphate nucleotides. These diphosphate nucleotides are not very good substrates for template directed reactions, but are chemically activated and are known to revert to the phosphoimidazolides in the presence of imidazole under solid state conditions. With respect to our studies of the oligomerization reaction, the determination of the dimerization rate constant of a specific ImpN (guanosine 5'-phospho 2 methylimidazolide) both in the absence and the presence of the template leads to the conclusion that at 37 C the dimerization is not template directed, although the subsequent polymerization steps are. In other words, this specific polynucleotide synthesizing system favors the elongation of oligonucleotides as compared with the formation of dimers and trimers. This favoring of the synthesis of long as opposed to short oligonucleotides may be regarded as a rudimentary example of natural selection at the molecular level.

Kanavarioti, A.↗
NASA NTRS1987

Non-enzymatic transcription of an oligodeoxynucleotide 14 residues long

Nonenzymatic synthesis of oligodeoxynucleotides up to 14 residues long from 2-MeImpG and 2-MeImpC mononucleotides was demonstrated. The synthesis is primed by 14-mer and 15-mer oligonucleotides d(C3GC3GC3GC2) and d(C3GC3GC3GC3) as templates. The predominant products are a series of 3-prime-5-prime-linked oligonucleotides, complementary to the template, ranging in length from GGC to GGCGGGCGGGCGGG. The 15-mer template directed the synthesis of the same family of products that were formed on the 14-mer template. This finding is explained by the preferential conversion of the dimer GG to GGC rather than to GGG. In the context of molecular evolution, these results suggest that the detailed kinetics of template-directed synthesis could form the basis for the selection of one replicating oligonucleotide from a family of closely related oligonucleotides.

Acevedo, Oscar L.↗
NASA NTRS1996

Synthesis of RNA oligomers on heterogeneous templates

The concept of an RNA world in the chemical origin of life is appealing, as nucleic acids are capable of both information storage and acting as templates that catalyse the synthesis of complementary molecules. Template-directed synthesis has been demonstrated for homogeneous oligonucleotides that, like natural nucleic acids, have 3',5' linkages between the nucleotide monomers. But it seems likely that prebiotic routes to RNA-like molecules would have produced heterogeneous molecules with various kinds of phosphodiester linkages and both linear and cyclic nucleotide chains. Here we show that such heterogeneity need be no obstacle to the templating of complementary molecules. Specifically, we show that heterogeneous oligocytidylates, formed by the montmorillonite clay-catalysed condensation of actuated monomers, can serve as templates for the synthesis of oligoguanylates. Furthermore, we show that oligocytidylates that are exclusively 2',5'-linked can also direct synthesis of oligoguanylates. Such heterogeneous templating reactions could have increased the diversity of the pool of protonucleic acids from which life ultimately emerged.

Non-NASA Center↗
NASA NTRS1979

Prebiotic nucleotide oligomerization in a fluctuating environment - Effects of kaolinite and cyanamide

The clay kaolinite was tested for its ability to promote nucleotide oligomerization in model prebiotic systems. Heterogeneous mixtures of clay, water and nucleotide were repeatedly evaporated to dryness at 60 C and redissolved in water in cyclic fashion in the presence or absence of cyanamide and/or ammonium chloride. With or without cycling, kaolinite alone did not promote the oligomerization of nucleotides at detectable levels. Cycling of clay in combination with cyanamide, however, promoted high levels of condensation to a mixture of oligonucleotides and dinucleotide pyrophosphate without requiring ammonium chloride. Although cycling with clay favored synthesis of dinucleotide pyrophosphate, cycling without clay enhanced formation of oligonucleotides. These results support the hypothesis that the presence of clays in fluctuating environments would have influenced the course of prebiotic condensation reactions.

Odom, D. G.↗
NASA NTRS1984

Nucleic acids, proteins, and chirality

The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

Usher, D. A.↗