We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA ("expanded DNA") and yDNA ("wide DNA"). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids.

Previous studies have explored whether it is possible to replace the information-encoding part of DNA-the bases and base pairs-with other molecular replacements, and whether such new base pairs might function in recognition and in replication processes, two of the defining characteristics of the natural genetic system. [1][2][3][4][5][6] Many of these studies have been aimed at designing base pairs that can function within the content of the natural genetic system, for expansion of natures genetic alphabet. However, recent studies have moved beyond the purine-pyrimidine framework of the natural system. Examples of this new approach include metal-bridged base pairs, [4] nonpolar base pairs, [2,4] and pairs containing more than the three different types of hydrogen bonds found in nature. [5] We recently adopted a different strategy and described a molecular design in which the dimensions of the natural pairs are stretched by insertion of a benzene ring into the natural heterocycles, thus rendering base pairs 2.4 wider than the natural ones.[6] Such broad alterations of the natural design are not necessarily expected to be compatible with the natural genetic system. Rather, such studies are useful as a test of researchers understanding of how genetic systems function in general, and they may also lead to useful new applications in biotechnology and in selfassembling nanostructures.Herein we evaluate whether size-expansion of DNA base pairs can be carried out using a new geometry. Our previous design of expanded DNA-like bases (xDNA) involved a linear extension of pyrimidines and purines by addition of a benzene ring to each of the four DNA base heterocycles. [6] However, examination of models suggested that at least one other design strategy involving benzohomologation also appeared to allow for reasonable base-pair geometries and stacking with neighboring pairs. This new design, termed "yDNA" (an abbreviated form of "wide DNA"; Scheme 1) involves a different extension vector, but yields a similar degree of perturbation from the framework formed by the natural pair. Analogous designs for the other three nucleobases can be envisioned (not shown). In this initial study we tested the concept with one example, that of the expanded adenine base analogue (yA) and the corresponding deoxyriboside (dyA), both of which were previously unknown.We developed a synthetic route to prepare the phosphoramidite derivative of the new nucleoside analogue dyA in 10 steps (6 % overall yield on a gram scale, Scheme 2). Methylation of indole 1 at the 5 position was achieved by addition of three equivalents of methylmagnesium chloride to the unprotected indole 1, followed by ring oxidation to restore aromaticity. [7] The transformation of methylindole 3 [8] to indole-5-carboxaldehyde 4 was a key step; tris(dimethylamino)methane converted 3 into the enamine intermediate which was then oxidized by KMnO 4 in one pot to afford compound 4.[9] The protected 2'-deoxyriboside 7 was formed in three subsequent steps from the indole-5-aldehyde 4. Conv...

The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated in Escherichia coli using two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC–MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.

Spectinabilin is a rare nitrophenyl-substituted polyketide metabolite. Here we report the cloning and heterologous expression of the spectinabilin gene cluster from Streptomyces spectabilis. Unexpectedly, this gene cluster is evolutionarily closer to the aureothin gene cluster than to the spectinabilin gene cluster from Streptomyces orinoci. Moreover, the two nearly identical spectinabilin gene clusters use a distinctly different regulation mechanism.

The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18–200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.