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ABSTRACT: Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.
Nature Chemical Biology 06/2012; 8(7):612-4. · 14.69 Impact Factor
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Chemistry 06/2011; 17(24):6606-9. · 5.93 Impact Factor
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ABSTRACT: As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue dMMO2, which forms an unnatural base pair with the nucleotide analogue d5SICS. Replacing the para-methyl substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically negative effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO-d5SICS, and especially dDMO-d5SICS, represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure-activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental dMMO2-d5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.
Chemistry 11/2010; 16(42):12650-9. · 5.93 Impact Factor
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ABSTRACT: 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. The results described here indicate that a sequence-specific DNA binding ligand such as actinomycin D will, indeed, recognize and bind with high affinity to a DNA incorporating a non-natural, non-hydrogen-bonding nucleoside mimic despite the presentation of modified functionality in the binding site.
Journal of the American Chemical Society 11/2010; · 9.91 Impact Factor
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ABSTRACT: The 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. A detailed structural analysis reveals that the absence of hydrogen bonding introduces more dynamic motion into the G-F pair relative to G-T and permits the G-F pair to exhibit stacking and conformational features characteristic of both a Watson-Crick base pair (on the guanine containing strand) and a wobble base pair (on the strand containing the difluorotoluene). We used these results to posit a rationale for recognition and repair of mismatch sites in DNA.
Journal of the American Chemical Society 05/2008; 130(14):4869-78. · 9.91 Impact Factor
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Shigeo Matsuda,
Jeremiah D Fillo,
Allison A Henry,
Priyamrada Rai,
Steven J Wilkens, Tammy J Dwyer,
Bernhard H Geierstanger,
David E Wemmer,
Peter G Schultz,
Glen Spraggon,
Floyd E Romesberg
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ABSTRACT: Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.
Journal of the American Chemical Society 09/2007; 129(34):10466-73. · 9.91 Impact Factor
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ABSTRACT: We used a facile, aqueous reaction coupled with gas chromatography–mass spectrometry (GC–MS), visible spectrophotometry, and high performance liquid chromatography (HPLC) to quantify the amounts of α-dicarbonyl compounds in wine samples. The aqueous reaction between an α-dicarbonyl compound and o-phenylenediamine yields a highly stable quinoxaline molecule with an absorbance maximum of 315 nm. GC–MS is used to identify each quinoxaline via mass. Likewise, owing to the similar absorption properties of the quinoxaline products, visible spectrometry is not useful for quantitation although it reveals the absorbance maximum to use for optical detection of the quinoxalines by HPLC. The analytical techniques used in this experiment complement each other since visible spectrophotometry is needed to determine the absorption wavelength, which is used for HPLC analysis; and HPLC is used for quantitation, while GC–MS is used to confirm the identities of the reaction products. Keywords (Audience): Second-Year Undergraduate
01/2006;
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ABSTRACT: The solution structure of a cyclic polyamide ligand complexed to a DNA oligomer, derived from NMR restrained molecular mechanics, is presented. The polyamide, cyclo-gamma-ImPyPy-gamma-PyPyPy-, binds to target DNA with a nanomolar dissociation constant as characterized by quantitative footprinting previously reported. 2D (1)H NMR data were used to generate distance restraints defining the structure of this cyclic polyamide with the DNA duplex d(5'-GCCTGTTAGCG-3'):d(5'-CGCTAACAGGC-3'). Data interpretation used complete relaxation matrix analysis of the NOESY cross-peak intensities with the program MARDIGRAS. The NMR-based distance restraints (276 total) were applied in restrained molecular dynamics calculations using a solvent model, yielding structures with an rmsd for the ligand and binding site of approximately 1 A. The resulting structures indicate some distortion of the DNA in the binding site. The constraints from cyclization lead to altered stacking of the rings in the halves of the cyclic ligand relative to unlinked complexes. Despite this, the interactions with DNA are very similar to what has been found in unlinked complexes. Measurements of ligand amide and DNA imino proton exchange rates indicate very slow dissociation of the ligand and show that the DNA can undergo opening fluctuations while the ligand is bound although the presence of the ligand decreases their frequency relative to the free DNA.
Journal of the American Chemical Society 07/2004; 126(25):7958-66. · 9.91 Impact Factor
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04/2002;
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04/2002;
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ABSTRACT: We are using Silicon Graphics workstations and mol. modeling software including Insight II/Discover (Mol. Simulations) and Spartan (Wavefunction, Inc.) to facilitate visualization of mol. and electronic structures and to help draw their connections to function and reactivity. The NSF-ILI funded workstations are being used in general chem. lab., org. chem. lab., phys. chem. lecture, two advanced integrated labs., biochem. lecture, and two biochem. labs. Examples to be discussed include 1) the development of tutorials that guide students through investigations of mol. structure, stereochem., reactivity, and structure/energy relationships; 2) the calcn. and display of min. energy conformations, MOs and electron d. surfaces; and 3) the development of a undergraduate special topics course in computational chem. [on SciFinder (R)]
Book of Abstracts, 216th ACS National Meeting, Boston, August 23-27.
