The solution structures of two different DNA duplexes (one containing a G-T mismatched base pair and the other a non-hydrogen-bonding G-F pair, where F is difluorotoluene) in complex with the peptide antibiotic actinomycin D (ActD) are presented. Using (1)H, (19)F NMR, and molecular dynamics simulations, we show that there are three major differences between the complexes: (1) ActD binds to the GF duplex in an orientation that is flipped 180° relative to its position in the GT duplex; (2) whereas the difluorotoluene moiety takes the typical anti glycosidic conformation in the "free" (uncomplexed) GF duplex, it takes the syn conformation in the GF:ActD complex; and (3) in GF:ActD, the difluorotoluene moiety is completely unstacked in the helix; however, the guanine of the G-F pair is stacked quite well with the ActD intercalator and the flanking base on the 5' side. In GT:ActD, the G-T base pair (although pushed into the major groove from the non-Watson-Crick hydrogen-bonding pattern) stacks favorably with the ActD intercalator and the flanking base pair on the 5' side.
View Article and Find Full Text PDFA fluorophore/quencher pair capable of detecting conformational changes of DNA-protein complexes is described. The system employs a fluorescent nucleoside analog 1,3-diaza-2-oxophenothiazine (tC) within duplex DNA and a non-fluorescent quencher (TEMPO) attached to an engineered cysteine residue of the protein. The straightforward labeling methodology allows for the placement of the fluorophore and quencher moieties at specific positions suited to studying the conformational change of interest.
View Article and Find Full Text PDFThe pathway leading to the preparation of a novel tricyclic 2'3'-dideoxycytosine analog, tCdd (1) is reported. A protected 2'3'-dideoxyribose prepared from l-glutamic acid was coupled to a silylated fluorescent base to yield a mixture of the alpha- and beta-anomers of the 2'3'-dideoxyribonucleoside of 1,3-diaza-2-oxophenothiazine, tCdd (1). The fluorescent base analog retains a high fluorescence emission over a large pH range and should be useful in a variety of probe applications.
View Article and Find Full Text PDFThe incorporation of synthetic nucleoside analogues into DNA duplexes provides a unique opportunity to probe both structure and function of nucleic acids. We used 1H and 19F NMR and molecular dynamics calculations to determine the solution structures of two similar DNA decamer duplexes, one containing a central G-T mismatched or "wobble" base pair, and one in which the thymine in this base pair is replaced by difluorotoluene (a thymine isostere) creating a G-F pair. Here, we show that the non-hydrogen-bonding G-F pair stacks relatively well into the helix and that the distortions caused by each non-Watson-Crick G-T or G-F base pair are quite localized to a three base pair site around the mismatch.
View Article and Find Full Text PDFWe describe the preparation and structure of the deoxyribonucleoside of 4-fluoro-6-methylbenzimidazole, abbreviated dH (8), which acts as a close shape mimic of the nucleoside deoxyguanosine. The nucleoside is prepared from 2-fluoro-4-methylaniline in seven steps. The X-ray crystal structure reveals a (-sc) glycosidic orientation, an S conformation for the deoxyribose moiety, and quite close shape mimicry of guanine by the substituted benzimidazole.
View Article and Find Full Text PDFAn organic trialdehyde, TRIPOD (2), was designed as a template for the synthesis of a three-helix bundle protein. Crystallographic data indicate that the aldehyde groups are appropriately spaced to maximize hydrophobic interactions between the chains of the protein. Peptide strands were attached to the template by reductive amination to yield a bundle protein that is 80% helical at pH 4.
View Article and Find Full Text PDFWe report the use of thermodynamic measurements in a self-complementary DNA duplex (5'-dXCGCGCG)(2), where X is an unpaired natural or nonnatural deoxynucleoside, to study the forces that stabilize aqueous aromatic stacking in the context of DNA. Thermal denaturation experiments show that the core duplex (lacking X) is formed with a free energy (37 °C) of -8.1 kcal·mol(-1) in a pH 7.
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