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With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined CE protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2'-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence, the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in ssDNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.

Saenger sequencing has led the advances in molecular biology, while faster and cheaper next generation technologies are urgently needed. A newer approach exploits nanopores, natural or solid-state, set in an electrical field, and obtains base sequence information from current variations due to the passage of a ssDNA molecule through the pore. A hurdle in this approach is the fact that the four bases are chemically comparable to each other which leads to small differences in current obstruction. 'Base calling' becomes even more challenging because most nanopores sense a short sequence and not individual bases. Perhaps sequencing DNA via nanopores would be more manageable, if only the bases were two, and chemically very different from each other; a sequence of 1s and 0s comes to mind. Osmylated DNA comes close to such a sequence of 1s and 0s. Osmylation is the addition of osmium tetroxide bipyridine across the C5-C6 double bond of the pyrimidines. Osmylation adds almost 400% mass to the reactive base, creates a sterically and electronically notably different molecule, labeled 1, compared to the unreactive purines, labeled 0. If osmylated DNA were successfully sequenced, the result would be a sequence of osmylated pyrimidines (1), and purines (0), and not of the actual nucleobases. To solve this problem we studied the osmylation reaction with short oligos and with M13mp18, a long ssDNA, developed a UV-vis assay to measure extent of osmylation, and designed two protocols. Protocol A uses mild conditions and yields osmylated thymidines (1), while leaving the other three bases (0) practically intact. Protocol B uses harsher conditions and effectively osmylates both pyrimidines, but not the purines. Applying these two protocols also to the complementary of the target polynucleotide yields a total of four osmylated strands that collectively could define the actual base sequence of the target DNA.

Polymeric compounds similar to oligonucleotides are relevant to the origin of life and particularly to the concept of an RNA world. Although short oligomers of RNA can be synthesized nonenzymatically under laboratory conditions by second-order reactions in concentrated solutions, there is no consensus on how these polymers could have been synthesized de novo on the early Earth from dilute solutions of monomers. To address this question in the context of an RNA world, we have explored ice eutectic phases as a reaction medium. When an aqueous solution freezes, the solutes become concentrated in the spaces between the ice crystals. The increased concentration offsets the effect of the lower temperature and accelerates the reaction. Here we show that in the presence of metal ions in dilute solutions, frozen samples of phosphoimidazolide-activated uridine react within days at -18 degrees C to form oligouridylates up to 11 bases long. Product yields typically exceed 90%, and approximately 30% of the oligomers include one or more 3'-5' linkages. These conditions facilitate not only the notoriously difficult oligouridylate synthesis, but also the oligomerization of activated cytidylate, adenylate, and guanylate. To our knowledge, this represents the first report to indicate that ice matrices on the early Earth may have accelerated certain prebiotic polymerization reactions.

Polycytidylate, poly(C), serves as a scaffold or template to direct and catalyze the synthesis of long oligoguanylates from guanosine 5'-phosphate 2-methylimidazolide, 2-MeImpG. In the absence of poly(C), small amounts of three isomeric dimers, i.e., the 2'-5'-, the 3'-5'-, and the pyrophosphate-linked, are formed slowly. In the presence of poly(C) oligomers that are primarily 3'-5'-linked are formed quickly and in high yield. Product analysis suggests that the oligomers are elongation products of the 3'-5'-linked dimer, abbreviated D. Assuming that D is formed slowly from two molecules of 2-MeImpG (Scheme 1) and elongates relatively fast, the initial rate of dimerization, d[D]/dt in M h(-1), was determined using two independent methods. The first method is based on the approximation that at the onset of the reaction the substrate is consumed only via hydrolysis and dimerization, and thus elongation can be neglected. The second, more accurate, method exploits the assertion that every oligomer was once a 3'-5'-linked dimer. Hence the concentration of D was obtained indirectly from the concentration of the oligomer products. These two methods gave comparable results. Experiments were run in aqueous solution in the presence of 1.0 M NaCl, 0.2 M MgCl(2) at pH 7.9 +/- 0.1 and 23 degrees C. Controls were run in the absence of poly(C) and in the presence of other polynucleotides. The kinetics were determined as a function of both monomer and polymer concentration the latter expressed in C equivalents. The kinetic data obtained in the presence of poly(C) confirmed an earlier conclusion regarding the remarkable effect of poly(C) on the formation of the 3'-5'-linked diguanylate. Initial dimerization rates were quantitatively correlated using a simple template-directed (TD) model that presumes cooperative binding (two association constants) of 2-MeImpG on poly(C) and reaction between adjacent template-bound molecules. The model allows for the estimation of the association constants and the intrinsic rate constant of dimerization, k(2). Insights into the detailed mechanism are also gained from this analysis. The fact that the proposed model can successfully correlate kinetic data that vary by more than 5000-fold between the slowest and the fastest reaction adds confidence and suggests the suitability of this model for describing TD reactions in general. It is anticipated that similar analysis of other known TD reactions may lead to clues that will facilitate the design of more efficient polynucleotide-synthesizing systems.

With the recent advances in electron microscopy (EM), computation, and nanofabrication, the original idea of reading DNA sequence directly from an image can now be tested. One approach is to develop heavy atom labels that can provide the contrast required for EM imaging. While evaluating tentative labels for the respective nucleobases in synthetic oligodeoxynucleotides (oligos), we developed a streamlined capillary electrophoresis (CE) protocol to assess the label stability, reactivity, and selectivity. We report our protocol using osmium tetroxide 2,2′-bipyridine (Osbipy) as a thymidine (T) specific label. The observed rates show that the labeling process is kinetically independent of both the oligo length, and the base composition. The conditions, i.e. temperature, optimal Osbipy concentration, and molar ratio of reagents, to promote 100% conversion of the starting oligo to labeled product were established. Hence the optimized conditions developed with the oligos could be leveraged to allow osmylation of effectively all Ts in single-stranded (ss) DNA, while achieving minimal mislabeling. In addition, the approach and methods employed here may be adapted to the evaluation of other prospective contrasting agents/labels to facilitate next-generation DNA sequencing by EM.

The hydrolysis of the title compound occurs by the four-step mechanism typical for the hydrolysis of olefins PhCH=CXY where X and/or Y are ele- ctron-withdrawing groups. These steps are nucleophilic attack by OH - or water on the olefin to form PhCH(OH)C(COMe) 2 (T - OH ); carbon pro- tonation of T - OH to form PhCH(OH)CH(COMe) 2 (T 0 OH ); oxygen depro- tonation of T 0 OH to form PhCH(O - )CH(COMe) 2 (T - O ); and collapse of T O - to PhCH=O and acetylacetonate anion. In strongly basic solution two kinetic processes were observed. To a good approximation the fast one can be assigned to reversible OH - addition to the substrate (k 1 - OH , k -1 H2 O ) to form T - OH and the slow one to irreversible con- version of T - OH to final products with rate-limiting carbon proto- nation of T - OH by water (k 2 H2 O ). In acidic solution only one kinetic process was observed; it corresponds to rate-limiting water addition to the substrate (k 1 H2 O )

A commonly accepted view is that life began in a marine environment, which would imply the presence of inorganic ions such as Na+, Cl-, Mg2+, Ca2+, and Fe2+. We have investigated two processes relevant to the origin of life--membrane self-assembly and RNA polymerization--and established that both are adversely affected by ionic solute concentrations much lower than those of contemporary oceans. In particular, monocarboxylic acid vesicles, which are plausible models of primitive membrane systems, are completely disrupted by low concentrations of divalent cations, such as magnesium and calcium, and by high sodium chloride concentrations as well. Similarly, a nonenzymatic, nontemplated polymerization of activated RNA monomers in ice/eutectic phases (in a solution of low initial ionic strength) yields oligomers with80% of the original monomers incorporated, but polymerization in initially higher ionic strength aqueous solutions is markedly inhibited. These observations suggest that cellular life may not have begun in a marine environment because the abundance of ionic inorganic solutes would have significantly inhibited the chemical and physical processes that lead to self-assembly of more complex molecular systems.

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.

Phosphoimidazolide activated ribomononucleotides (*pN) are useful substrates for the non-enzymatic synthesis of polynucleotides. However, dilute neutral aqueous solutions of *pN typically yield small amounts of dimers and traces of polymers; most of *pN hydrolyzes to yield nucleoside 5'-monophosphate. Here we report the self-condensation of nucleoside 5'-phosphate 2-methylimidazolide (2-MeImpN with N = cytidine, uridine or guanosine) in the presence of Mg2+ in concentrated solutions, such as might have been found in an evaporating lagoon on prebiotic Earth. The product distribution indicates that oligomerization is favored at the expense of hydrolysis. At 1.0 M, 2-MeImpU and 2-MeImpC produce about 65% of oligomers including 4% of the 3',5'-linked dimer. Examination of the product distribution of the three isomeric dimers in a self-condensation allows identification of reaction pathways that lead to dimer formation. Condensations in a concentrated mixture of all three nucleotides (U,C,G mixtures) is made possible by the enhanced solubility of 2-MeImpG in such mixtures. Although percent yield of internucleotide linked dimers is enhanced as a function of initial monomer concentration, pyrophosphate dimer yields remain practically unchanged at about 20% for 2-MeImpU, 16% for 2-MeImpC and 25% of the total pyrophosphate in the U,C,G mixtures. The efficiency by which oligomers are produced in these concentrated solutions makes the evaporating lagoon scenario a potentially interesting medium for the prebiotic synthesis of dimers and short RNAs.

We have studied the hydrolysis of guanosine 5'-phospho-2-methylimidazolide, 2-MeImpG, in aqueous buffered solutions of various pH's at 75 degrees C and 37 degrees C. At 75 degrees C and pHor = 1.0, two kinetic processes were observed spectrophotometrically: the first and more rapid one is attributed to the hydrolysis of the phosphoimidazolide P-N bond; the second and much slower one, to the cleavage of the glycosidic bond. At 37 degrees C, pH 2.0, the spectrophotometrically determined rate constant of P-N bond hydrolysis was confirmed by using high pressure liquid chromatography, HPLC. With the latter technique it was possible to separate reactants and products and also to extend the pH-rate profile into the neutral region where rates are slower and, therefore, difficult to measure spectrophotometrically. The pH-rate profiles at both temperatures exhibit similar behavior. At pH2 the pseudo-first-order rate constant increases with decreasing pH; in the region 2pH7 there is a plateau followed by a decrease for pH7. These data are consistent with a reactivity order zwitterionanion for P-N bond hydrolysis. It is noteworthy that P-N bond hydrolysis in phosphoimidazolides is very slow compared to other phosphoramidates. This may be one of the reasons why this compound showed extraordinary ability in forming long oligomers under template-directed conditions.