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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.

Symmetrical dinucleoside 5'-pyrophosphates have been synthesized from the corresponding nucleoside 5'-phosphate free acid in high yield. The one-pot procedure is carried out in DMF or DMSO using triphenylphosphine and 2,2'-dipyridyldisulfide as the coupling agents, and 1-methylimidazole as the catalyst.

The formation of the internucleotide bond in diguanylate synthesis was studied in aqueous solution at pH 8 and 0.2 M Mg2+ in the presence and absence of polycytidylate, poly(C). The investigation was simplified by using guanosine 5'-phosphorylmorpholinamide, mor-pG, which can act only as a nucleophile, and deoxyguanosine 5'-phosphoryl-2-methylimidazolide, 2-MeImpdG, which can act only as an electrophile. The time-dependent product distribution was monitored by high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC/MS). In the absence of poly(C) the reaction between mor-pG and 2-MeImpdG yielded small amounts of the dimer mor-pGpdG with a regioselectivity of 2'-5':3'-5' = 3.5. In the presence of poly(C) dimer yields increased and a reversal in regioselectivity occurred; both effects were in proportion to the concentration of the polymer. The results can be quantitatively explained with the proposition that poly(C), acting as the template, catalyzes the reaction between template-bound monomers by about a factor of 4-5 over the reaction in solution and yields dimers with a regioselectivity of 2'-5':3'-5' approximately 0.33. These findings illustrate the intrinsic preference of guanosine monomers to correctly self-assemble on the appropriate template.

A detailed investigation of the hydrolysis of (Ia) in the pH range 6-12.4 at 37 °C is presented.