2013

  • Expanding the scope of replicable unnatural DNA: Stepwise optimization of a predominantly hydrophobic base pair

    T. Lavergne, M. Degardin, D.A. Malyshev, H.T. Quach, K. Dhami, P. Ordoukhanian, F.E. Romesberg, J. Am. Chem. Soc. Accepted for publication 12 March 2013

    Here, we report the stepwise optimization of dMMO2 via the synthesis and evaluation of eighteen novel para-derivatized analogs of dMMO2, followed by further derivatization and evaluation of the most promising analogs with meta substituents. The most promising, d5SICS-dFEMO, is replicated under some conditions with greater efficiency and fidelity than d5SICS-dNaM.

2012

  • Efficient and sequence-independent replication of DNA containing a third base pair establishes a functional six-letter genetic alphabet

    D.A. Malyshev, K. Dhami, H.T. Quach, T. Laveryne, P. Ordoukhanian, A. Torkamani, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2012) 109:12005-12010.

    We examine the PCR amplification of DNA containing one or more d5SICS-dNaM pairs and demonstrate that this DNA may be amplified with high efficiency and with greater than 99.9% fidelity. The results demonstrate that for PCR and PCR-based applications, d5SICS-ddNaM is functionally equivalent to a natural base pair, and when combined with dA-dT and dG-dC, provides the first fully functional six-letter genetic alphabet.

  • The mechanism of action of the arylomycin antibiotics and the effects of signal peptidase I inhibition

    P.A. Smith, F.E. Romesberg, Antimicrob. Agents Chemother. (2012) 56:5054-5060.

    We examine arylomycin activity as a function of concentration, bacterial cell density, target expression levels, and bacterial growth phase. We find that the activity of the arylomycins results from an insufficient flux of proteins through the secretion pathway and the resulting mislocalization of proteins. We also demonstrate that SPase inhibition results in synergistic sensitivity when combined with an aminoglycoside.

  • Protein dynamics and the diversity of the antibody response

    R. Adhikary, W. Yu, M. Oda, J. Zimmermann, F.E. Romesberg, J. Biol. Chem. (2012) 287:27139-27147.

    We report the sequence, thermodynamic, and time-resolved spectroscopic characterization of a panel of eight antibodies (Abs) elicited to a chromophoric antigen (MPTS). Three of the Abs arose from unique germline Abs, while the remaining five comprise two sets of siblings that arose by somatic mutation of a common precursor.By characterizing both flexibility and conformational heterogeneity, we show that point mutations are capable of fixing significant differences in protein dynamics.

  • Yeasts acquire resistance secondary to antifungal drug treatment by adaptive mutagenesis

    D. Quinto-Alemany, A. Canerina-Amaro, L.G. Hernández-Abad, F. Machín, F.E. Romesberg, C. Gil-Lamaignere, PLoS ONE (2012) 7:e42279.

    Stressful lifestyle associated mutation (SLAM) assays are used to demonstrate that both S. cerevisiae and C. albicans undergo mutations to acquire resistance to the antifungal agents 5-fluorocytosine and caspofungin. Examination of the mutation spectrum shows that these aquired mutations are different from those that the occur spontaneously, in the absence of antifungal treatment. Overall, the work present a much needed model system for studying adaptive mutagenesis in eukaryotes.

  • KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

    K. Betz, D.A. Malyshev, T. Lavergne, W. Welte, K. Diederichs, T.J. Dwyer, P. Ordoukhanian, F.E. Romesberg, A. Marx, Nat. Chem. Biol. (2012) 8:612-614

    We report crystal structures of KlenTaq DNA polymerase at different stages of replicating dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.

  • Carbon-deuterium bonds as probes of protein thermal unfolding

    W. Yu, P. Dawson, J. Zimmermann, F.E. Romesberg, J. Phys. Chem. B (2012) 116:6397-6403.

    We report a residue-specific characterization of the thermal unfolding mechanism of cytochrome c using C-D bonds site-specifically incorporated at residues dispersed throughout three different structural elements within the protein. Elucidation of the detailed unfolding mechanism and the structure of the associated molten globule, both of which represent challenges to conventional techniques, are highlights of the utility of the C-D technique.

  • REVIEW: Latent antibiotics and the potential of the arylomycins for broad-spectrum antibacterial activity

    Y.X. Tan, F.E. Romesberg, MedChemComm (2012) 3:916-925.

    We review the arylomycin family of natural product antibiotics, highlighting how their characterization has provided new insight into how antibiotics evolve in nature.

  • Type I signal peptidase and protein secretion in S. aureus

    M.A. Schallenberger, S. Niessen, C. Shao, B.J. Fowler, F.E. Romesberg, J. Bacteriol. (2012) 194:2677-2686

    We use an arylomycin as a probe of protein secretion to identify forty-six proteins whose extracellular accumulation in S. aureus requires the activity of signal peptidase (SPase). These include a wide variety of virulence proteins with identifiable Sec-type signal peptides, as well as some proteins with non-canonical cleavage sites.

  • Major groove substituents and polymerase recognition of a class of unnatural base pairs

    T. Lavergne, D.A. Malyshev, F.E. Romesberg, Chem. Eur. J. (2012) 18:1231-1239.

    The continued optimization of a predominantly hydrophobic class of unnatural base pairs is reported. We find that minimizing the cost of nucleobase desolvation and optimizing packing interactions within the developing major groove of DNA is promising route toward optimization. While we identify an improved base pair, dNMO1-d5SICS, continued analysis of the previously reported dNaM-d5SICS pair reveals that it is replicated with an efficiency and fidelity approaching those of natural DNA.

2011

  • Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs

    Y.J. Seo, D.A. Malyshev, T. Lavergne, P. Ordoukhanian, F.E. Romesberg, J. Am. Chem. Soc. (2011) 133:19878-19888.

    We report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the post-amplification site-specific labeling of DNA.

  • Synthesis and characterization of the arylomycin lipoglycopeptide antibiotics and the crystallographic analysis of their complex with signal peptidase

    J. Liu, C. Luo, P.A. Smith, J.K. Chin, M.G.P. Page, M. Paetzel, F.E. Romesberg, J. Am. Chem. Soc., (2011) 133:17869-17877.

    We report the first total synthesis of a lipoglycopeptide member of the arylomycin family of natural products, and we demonstrate that it effectively penetrates Gram-negative bacteria and is limited by the same resistance mechanism as other members of the arylomycin class of antibiotics (i.e. mutation in the target protein signal peptidase (SPase)). Unlike the other arylomycins, the lipoglycopeptides are glycosylated, and the structural data reveal that this sugar is most solvent exposed when in complex with SPase.

  • Synthesis and biological evaluation of 2',4'- and 3',4'-bridged nucleoside analogues

    K.C. Nicolaou, S.P. Ellery, F. Rivas, K. Saye, E. Rogers, T.J. Workinger, M. Schallenberger, R. Tawatao, A. Montero, A. Hessell, F. Romesberg, D. Carson, D. Burton Bioor. Med. Chem. (2011) 19:5648-5669.

    A small library of 2′,4′- and 3′,4′-bridged nucleoside analogues was synthesized and tested for antibacterial, antitumor, and antiviral activities, leading to the identification of one nucleoside that exhibited significant antiviral activity against pseudoviruses SF162 (IC50 = 7.0 μM) and HxB2 (IC50 = 2.4 μM). These findings render bridged nucleosides as credible leads for drug discovery in the anti-HIV area of research.

  • Cyano groups as probes of protein microenvironments and dynamics

    J. Zimmermann, M.C. Thielges, Y.J. Seo, P.E. Dawson, F.E. Romesberg, Angew. Chem. Int. Ed. (2011) 50:8333-8337.

    The cyano group is sensitive to its environment, absorbs in a unique region of the protein IR spectrum, and may be appended to an amino acid. Using both steady-state and time-resolved methods, we explore the use of cyano groups as probes of protein microenvironments and dynamics in variants of cytochrome c. We find that the cyano group is a useful site-specific probe of protein microenvironments and dynamics, but that it can also perturb its environment and destabilize the folded state of the protein.

  • Initial efforts toward the optimization of arylomycins for antibiotic activity

    T.C. Roberts, M. Schallenberger, J. Liu, P.A. Smith, F.E. Romesberg, J. Med. Chem. (2011) 54:4954-4963.

    Resistance to the arylomycins in several key human pathogens is due to the presence of a specific Pro residue in the target peptidase that disrupts interactions with the lipopeptide tail of the antibiotic. Here, we describe the synthesis and characterization of arylomycin derivatives with altered lipopeptide tails, including several with an increased spectrum of activity against S. aureus.

  • Synthesis and biological characterization of arylomycin B antibiotics

    T.C. Roberts, P.A. Smith, F.E. Romesberg, J. Nat. Prod. (2011) 74:956-961.

    We report the total synthesis of arylomycin B-C16, and its aromatic amine derivative. While the aromatic amine loses activity against all bacteria tested, the B series compound shows activities that are similar to the A series compounds, except that it also gains activity against the important pathogen Streptococcus agalactiae.

  • Carbon-deuterium bonds as site-specific and non-perturbative probes for time-resolved studies of protein dynamics and folding

    J. Zimmermann, M.C. Thielges, W. Yu, P.E. Dawson, F.E. Romesberg, J. Phys. Chem. Lett. (2011) 2:412-416.

    We explore the use of carbon-deuterium (C-D) bonds under time-resolved conditions to follow the unfolding of cytochrome c from a photostationary state that accumulates after CO is photodissociated from the protein’s heme prosthetic group. Our results clearly show that C-D bonds are well-suited to characterize protein folding and dynamics.

  • In vitro activity of the arylomycin natural product antibiotics against Staphylococcus epidermidis and other coagulase-negative staphylococci

    P.A. Smith, M.E. Powers, T.C. Roberts, F.E. Romesberg, Antimicrob. Agents Chemother. (2011) 55:1130-1134.

    We report the activity of a synthetic arylomycin derivative against clinical isolates of coagulase-negative staphylococci (CoNS). Against many important CoNS, e.g. S. epidermidis, S. haemolyticus, S. lugdunensis, and S. hominis, we find that the arylomycin has activity equal to or greater than that of vancomycin, the antibiotic most commonly used to treat CoNS infections.

  • Type I Signal Peptidase and Protein Secretion in S. epidermidis

    M.E. Powers, P.A. Smith, T.C. Roberts, B.J. Fowler, C.C. King, S.A. Trauger, G. Siuzdak, F.E. Romesberg, J. Bacteriol. (2011) 193:340-348.

    This work describes the use of an arylomycin derivative, along with two-dimensional gel electrophoresis and LC-MS/MS, to identify eleven proteins whose secretion from stationary phase S. epidermidis is dependent on SPase activity. The data also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

2010

  • Broad spectrum antibiotic activity of the arylomycin natural products is masked by natural target mutations

    P.A. Smith, T.R. Roberts, F.E. Romesberg, Chem. Biol. (2010) 17:1223-1231.

    This work demonstrates that the apparently narrow spectrum of the arylomycin natural products results not from intrinsic limitations, but from target mutations and that the arylomycins have a much broader spectrum of activity than previously thought.

  • The psychotrimine natural products have antibacterial activity against Gram-positive bacteria and act via membrane disruption

    M.A. Schallenberger, T. Newhouse, P.S. Baran, F.E. Romesberg, J. Antibiot. (2010) 63:685-687.

    Plants provide a unique source of diverse secondary metabolites with potentially important biological activities, with one of the most promising classes being the indole alkaloids. One such indole alkaloid natural product is psychotrimine, which has attracted considerable interest from the synthetic and medicinal chemistry communities owing to its unusual connectivity between tryptamine subunits. Here, we examine the potential antibacterial activity of this unique indole alkaloid.

  • Secretion of the chlamydial virulence factor CPAF requires Sec-dependent pathway

    D. Chen, L. Lei, C. Lu, R. Flores, M.P. DeLisa, T.C. Roberts, F.E. Romesberg, G. Zhong, Microbiology (2010) 156:3031-3040.

    CPAF is secreted into host cytosol to degrade various host factors for benefiting chlamydial intracellular survival. Here, we show experimental evidence that the chlamydial virulence factor CPAF relies on sec-dependent transport for crossing the chlamydial organism inner membrane.

  • Solution structure, mechanism of replication, and optimization of an unnatural base pair

    D.A. Malyshev, D.A. Pfaff, S.I. Ippoliti, G.T. Hwang, T.J. Dwyer, F.E. Romesberg, Chem. Eur. J. (2010) 16:12650-12659.

    Here, we synthesize a variety of derivatized unnatural base pairs and characterize their structures and DNA polymerase-mediated replication. We also present further evidence supporting an intercalation-based mechanism of replication, and show that dDMO—d5SICS represents significant progresstoward the expansion of the genetic alphabet

  • Directed evolution of DNA polymerases for next generation sequencing

    A.M. Leconte, M.P. Patel, L.E. Sass, P. McInerney, M. Jarosz, L. Kung, J.L. Bowers, P.R. Buzby, J.W. Efcavitch, F.E. Romesberg, Angew. Chem. Int. Ed. (2010) 34:5921-5924.

    We identified a mutant of Taq DNA polymerase that incorporates fluorophore-labeled substrates 50 to 400-fold more efficiently into scarred primers in solution and also demonstrates significantly improved performance under actual sequencing conditions.

  • SOS regulatory elements are essential for UPEC pathogenesis

    B. Li, P. Smith, D.J. Horvath, Jr, F.E. Romesberg, S.S. Justice, Microb. Infect. (2010) 12:662-668.

    Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells.

  • A molecular description of flexibility in an antibody combining site

    J. Zimmermann, F.E. Romesberg, C.L. Brooks III, I.F. Thorpe, J. Phys. Chem. B (2010) 114:7359-7370.

    We show that 3PEPS spectroscopy in combination with molecular dynamics simulations can provide a detailed description of protein dynamics for the fluorescein Ab 4−4−20, and how it is evolved for biological function.

  • The folding energy landscape and free energy excitations of cytochrome

    P. Weinkam, J. Zimmermann, F.E. Romesberg, P. Wolynes, Acc. Chem. Res. (2010) 43:652-660.

    At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. We account for these alternative folding pathways using complex models that begin from native structure-based terms and also add the chemical frustration that arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH.

2009

  • PCR with an expanded genetic alphabet

    D. Malyshev, Y.J. Seo, P. Ordoukhanian, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:14620-14621.

    We demonstrate that DNA containing either the d5SICS−dNaM or d55SICS−dMMO2 unnatural base pair may be amplified by PCR with fidelities and efficiencies that approach those of fully natural DNA.

  • Major groove derivatization of an unnatural base pair

    Y.J. Seo, F.E. Romesberg, ChemBioChem (2009) 10:2394-2400.

    Cover Story

    We synthesize and evaluate several dMMO2 derivatives with meta-chlorine, -bromine, -iodine, -methyl, or -propinyl substituents. Complete characterization of unnatural base pair and mispair synthesis and extension reveal that the modifications have large effects only on the efficiency of unnatural base pair synthesis and that the effects likely result from a combination of changes in steric interactions, polarity, and polarizability.

  • The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation

    G.T. Hwang , Y. Hari, F.E. Romesberg, Nucleic Acids Res, (2009) 37:4757-4763.

    We examine six pyridine-based nucleotides, differentiated by methyl substitution, that are designed to vary both inter- and intra-strand packing within duplex DNA. We show that incorporation of one of these nucleobases, d34DMPy, as a self pair or as a mispair with dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex.

  • Efforts toward developing probes of protein dynamics: vibrational dephasing and relaxation of carbon-deuterium stretching modes in deuterated leucine

    J. Zimmermann, K. Gundogdu, M.E. Cremeens, J.N. Bandaria, G.T. Hwang, M.C. Thielges, C.M. Cheatum, F.E. Romesberg, J. Phys. Chem. B (2009) 113:7991-7994.

    We present the first time-resolved experiments characterizing the population and dephasing dynamics of selectively excited C−D bonds in a deuterated amino acid using both three-pulse photon echo and transient grating experiments. We show that the steady-state C−D absorption linewidths in the deuterated amino acid are broadened by both homogeneous and inhomogeneous effects, and that IVR occurs on a subpicosecond time scale and is nonstatistical.

  • Synthesis and biological evaluation of penem inhibitors of bacterial signal peptidases.

    D.A. Harris, M.E. Powers, F.E. Romesberg, Bioorg. Med. Chem. Lett. (2009) 19:3787-3790.

    We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid, and we demonstrate the first in vivo activity of the compound and use structure–activity relationship studies to begin to define the determinants of signal peptidase binding and to begin to optimize the penem as an antibiotic.

  • Direct observation of structural heterogeneity in a β-sheet

    M.E. Cremeens, J. Zimmermann, W. Yu, P.E. Dawson, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:5726-5727.

    We demonstrate that deuterium atoms incorporated at Cα backbone positions (Cα−D bonds) are sensitive to the local backbone structure. DFT calculations are used to predict that Cα−D bonds of glycine are sensitive to their local structure, with the absorptions red-shifted for an extended β-sheet relative to γ- and α-helix-like turns. These predictions are confirmed in glycine-deuterated variants of the N-terminal Src homology 3 (nSH3) domain protein.

  • Direct observation of ligand dynamics in cytochrome c

    M.T. Thielges, J. Zimmermann, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:6054-6055.

    We address the processes that occur upon folding of reduced cyt c induced by photodissociation of CO from the CO-bound, unfolded protein by monitoring the CO vibration with step-scan FT-IR spectroscopy.

  • Transcription of an expanded genetic alphabet

    Y.J. Seo, S. Matsuda, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:5046-5047.

    We show here that both of the unnatural base pairs d5SICS:dMMO2 and d5SICS:dNaM are selectively transcribed by T7 RNA polymerase and that the efficiency of d5SICS:dNaM transcription in both possible strand contexts is only marginally reduced relative to that of a natural base pair.

  • The determinants of stability and folding in evolutionarily diverged cytochromes c

    M.C. Thielges, J. Zimmermann, P.E. Dawson, F.E. Romesberg, J. Mol. Biol. (2009) 24:159-167.

    Using site-selectively incorporated carbon–deuterium bonds, we show that like equine cyt c, bovine cyt c is induced to unfold by guanidine hydrochloride via a stepwise mechanism, but it does not populate an intermediate as is observed with the equine protein.

  • Efforts toward the direct experimental characterization of enzyme microenvironments: tyrosine100 in dihydrofolate reductase

    D. Groff, M.C. Thielges, S. Celliti, P.G. Schultz, F.E. Romesberg, Angew. Chem., Int., Ed., (2009) 48:3478-3481.

    Site-specific deuteration and FT-IR studies reveal that Tyr100 in DHFR plays an important role in catalysis, with a strong electrostatic coupling occuring between Tyr100 and the charge that develops in the hydride-transfer transition state.

  • Chemical frustration in the protein folding landscape: grand canonical ensemble simluations of cytochrome c

    P. Weinkam, F.E. Romesberg, P.G. Wolynes, Biochemistry (2009) 48:2394-2402.

    A grand canonical formalism is developed to combine discrete simulations for chemically distinct species in equilibrium. Each simulation is based on a perturbed funneled landscape. The formalism is illustrated using the alkaline-induced transitions of cyt c as observed by FT-IR spectroscopy and with various other experimental approaches

  • C-D Modes of deuterated side chain of leucine as structural reporters via dual-frequency two-dimensional infrared spectroscopy

    S.R.G. Naraharisetty, V.M. Kasyanenko, J. Zimmermann, M.C. Thielges, F.E. Romesberg, I.V. Rubtsov, J. Phys. Chem B, (2009) 113:4940-4946.

    We demonstrate that the C-D transitions of the deuterated amino acid, leucine-d10 can serve as convenient structural reporters via dual-frequency 2DIR methods, and we discuss the potential of leucine-d10 and other amino acids with deuterium-labeled side chains as probes of protein structure and dynamics.

  • Optimization of an unnatural base pair towards natural-like replication

    Y.J. Seo, G.T. Hwang, P. Ordoukhanian, F.E. Romesberg, J. Am. Chem. Soc. (2009) 131:3246-3252.

    To better understand and optimize the slowest step of replication of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS, the insertion of MMO2 opposite d5SICS, we synthesize two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability, and we characterize their enzymatic replication.

  • Protein dynamics and the evolution of novel protein functions

    J. Zimmermann, M.C. Thielges, W. Yu, F.E. Romesberg, in Protein Engineering Handbook, Vol. 1 (2009) S. Lutz and U.T. Bornscheuer, Eds., Wiley-VCH:Weinheim, 147-186.

    We review novel experimental methods to characterize conformational heterogeneity and dynamics in proteins.

  • Engineering nucleobases and polymerases for an expanded genetic alphabet

    A.M. Leconte , F.E. Romesberg, in Protein Engineering (2009) C. Köhrer and U.L. RajBhandary, Eds., Springer-Verlag:Berlin, 291-314.

    We review screening and selection methods to identify polymerases with novel function.

2008

  • Characterization of alkaline transitions in ferricytochrome c using carbon-deuterium infrared probes

    P. Weinkam, J. Zimmermann, L.B. Sagle, S. Matsuda, P.E. Dawson, P.G. Wolynes, F.E. Romesberg, Biochemistry (2008) 47:13470-13480.

    We use acombination of experiments and simulations to provide a high-resolution view of the structural changes in cyt c with increasing alkaline conditions. Alkaline-induced transitions were characterized under equilibrium conditions by monitoring IR absorptions of carbon−deuterium chromophores incorporated at Leu68, Lys72, Lys73, Lys79, and Met80. The data suggest that at least four intermediates are formed as the pH is increased prior to complete unfolding of the protein.

  • Unnatural substrate repertoire of A, B, and X family DNA polymerases

    G.T. Hwang, F.E. Romesberg, J. Am. Chem. Soc. (2008) 130:14872-14882.

    We examine the ability of five different polymerases (family A polymerases from bacteriophage T7 and Thermus aquaticus, family B polymerases from Thermococcus litoralis and Thermococcus 9°N-7, and the family X polymerase, human polymerase β.) to replicate DNA containing four different unnatural nucleotides bearing predominantly hydrophobic nucleobase analogs, and we identify some aspects of recognition that are conserved.

  • Optimization of the pyridyl nucleobase scaffold for polymerase recognition and unnatural base pair replication

    Y. Hari, G.T. Hwang, A.M. Leconte, N. Joubert, M. Hocek, F.E. Romesberg, ChemBioChem, (2008) 9:2796-2799.

    We demonstrate that the pyridyl nucleobase scaffold can be optimized for replication, both as a self pair and as a component of a heteropair, and we identify a pyridyl-nucleotide self pair that is well recognized by a DNA polymerase, allowing it to be replicated and used to synthesize site-specifically labeled DNA in good yields.

  • Editorial overview: Next generation therapeutics

    A. Mapp , F.E. Romesberg, Curr. Opin. Chem. Biol. (2008) 12:387-388.

    Editorial comment for the special issue of Current Opinion in Chemical Biology describing several novel approaches to developing therapeutics that target both bacterial and human proteins.

  • Exploring the energy landscape of antibody-antigen complexes: protein dynamics, flexibility, and molecular recognition

    M.C. Thielges, J. Zimmermann, W. Yu, M. Oda, F.E. Romesberg, Biochemistry (2008) 47:7237-7247. [June 2008 Hot Article]

    We examine a panel of antibodies elicited to the chromophoric antigen fluorescein. On the basis of isothermal titration calorimetry, we select six antibodies that bind fluorescein with diverse binding entropies, suggestive of varying contributions of dynamics to molecular recognition. We find that more than half of all the somatic mutations among the six antibodies are located in positions unlikely to contact fluorescein directly, and using 3PEPS and TG spectroscopy, we find a high level of dynamic diversity among the antibodies.

  • Carbon-Deuterium bonds as probes of dihydrofolate reductase

    M.C. Thielges, D.A. Case, F.E. Romesberg, J. Am. Chem. Soc. (2008) 130:6597-6603.

    We report a preliminary analysis of (methyl-d3) methionine residues, Met16, Met20, and Met42, within DHFR. The results confirm the sensitivity of the carbon−deuterium bonds to their local protein environment and demonstrate that dihydrofolate reductase is electrostatically and dynamically heterogeneous.

  • Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

    E.T. Lis, B.M. O'Neill, C. Gil-Lemaignere, J.K. Chin, F.E. Romesberg, DNA Repair (2008) 7:801-810.

    To further understand the mutagenic response to DNA damage, we screen a collection of 4848 haploid gene deletion strains of S. cerevisiae for decreased damage-induced mutation of the CAN1 gene, and we identify a pathway of induced mutation that involves a replicative polymerase and that is effectively inhibited by the RNR inhibitor, hydroxyurea.

  • Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet

    A.M. Leconte, G.T. Hwang, S. Matsuda, P. Capek, Y. Hari, F.E. Romesberg, J. Am. Chem. Soc. (2008) 130:2336-2343.

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2007

  • Phosphatases, DNA Damage Checkpoints and Checkpoint Deactivation

    J. Heideker, E.T. Lis, F.E. Romesberg, Cell Cycle (2007) 6:3058-3064.

    We review progress toward understanding the functions of phosphatases in checkpoint deactivation in S. cerevisiae, focusing on the non-redundant roles of the type 2A phosphatase Pph3 and the PP2C phosphatases Ptc2 and Ptc3 in the deactivation of Rad53.

  • Structural and initial biological analysis of synthetic arylomycin A2

    T.C. Roberts, P.A. Smith, R.T. Cirz, F.E. Romesberg, J. Am. Chem. Soc. (2007) 129:15830-15838

    We report the synthesis and characterization of arylomycin natural products.

  • REVIEW: Controlling mutation: intervening in evolution as a therapeutic strategy

    R.T. Cirz and F.E. Romesberg, Crit. Rev. Biochem. Mol. Biol. (2007) 42:341-354.

    We review what is known about induced mutagenesis in bacteria as well as evidence that it contributes to the evolution of antibiotic resistance and we discuss the possibility that components of induced mutation pathways might be targeted for inhibition as a novel therapeutic strategy to prevent the evolution of antibiotic resistance.

  • REVIEW: Combating bacteria and drug-resistance by inhibiting mechanisms of persistence and adaptation

    P.A. Smith and F.E. Romesberg, Nat. Chem. Biol. (2007) 3:549-556.

    An improved understanding of bacterial stress responses and evolution suggests that in the ability of bacteria to survive antibiotic therapy either by transiently tolerating antibiotics or by evolving resistance may require specific biochemical processes. We review early efforts toward inhibiting these processes as a means to prolong the efficacy of current antibiotics and provide an alternative to escalating the current arms race between antibiotics and bacterial resistance.

  • Polymerase recognition and stability of fluoro substituted pyridone nucleobase analogs

    G.T. Hwang, A.M. Leconte, F.E. Romesberg, ChemBioChem. (2007) 8:1606-1611.

    We synthesize and characterize three fluoro-substituted pyridone nucleoside analogues. Generally, we find that the specific fluorine substitution pattern of the analogues has little impact on unnatural pair or mispair stability, but does significantly increase the rate at which the pyridone-based unnatural base pairs are extended.

  • Efforts toward expansion of the genetic alphabet: Structure and replication of unnatural base pairs

    S. Matsuda, J.D. Fillo, A.A. Henry, P. Rai, S. J. Wilkens, T.J. Dwyer, B.H. Geierstanger, D.E. Wemmer, P.G. Schultz, G. Spraggon, F.E. Romesberg,J. Am Chem. Soc. (2007) 129:10466-10473.

    We examine the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of the dPICS nucleobases 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.

  • AID-initiated purposeful mutations in immunoglobulin genes

    M.F. Goodman, M.D. Schaarf, F.E. Romesberg, Adv. Immunol. (2007) 94:127-155.

    We review the role of activation-induced cytidine deaminase (AID) in the complex process of somatic hypermutation (SHM) and present recent advances in experimental methods to characterize antibody dynamics as a function of SHM to help elucidate the role that the AID-induced mutations play in tailoring molecular recognition.

  • Psy2 and Pph3 form a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage

    B.M. O'Neill, S.J. Szyjka, E.T. Lis, A.O. Bailey, J.R. Yates III, O.M. Aparicio, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2007) 104:9290-9295.

    We present genetic and biochemical evidence that the type 2A-like protein phosphatase Pph3 forms a complex with Psy2 (Pph3–Psy2) that binds and dephosphorylates activated Rad53 during treatment with, and recovery from, methylmethane sulfonate-mediated DNA damage. Ourfindings suggest that Rad53 regulates replication fork restart and initiation of late firing origins independently and that regulation of these processes is mediated by specific Rad53 phosphatases.

  • Minor groove hydrogen bonds and the replication of unnatural base pairs

    S. Matsuda, A.M. Leconte, F.E. Romesberg, J. Am. Chem. Soc. (2007) 129:5551-5557.

    We examine the enzymatic synthesis of DNA with six different unnatural nucleotides bearing methoxy-derivatized nucleobase analogues. Different nucleobase substitution patterns were used to systematically alter the nucleobase electronics, sterics, and hydrogen-bonding potential. Our results indicate that ortho methoxy groups should be generally useful for the effort to expand the genetic alphabet.

  • The complete and SOS-mediated response of Staphylococcus aureus to the antibiotic ciprofloxacin

    R.T. Cirz, M.B. Jones, N.A. Gingles, T.D. Minogue, B. Jarrahi, S.N. Peterson, F.E. Romesberg, J. Bacteriol. (2007) 189:531-539.

    We characterize the global transcriptional response of S. aureus to ciprofloxacin and find that the drug induces prophage mobilization as well as significant alterations in metabolism, most notably the up-regulation of the tricarboxylic acid cycle.[Download Supporting Information]

2006

  • From therapeutic nucleic acids to redox hydrogels: the diverse world of biopolymers

    H. Bayley, F.E. Romesberg, Curr. Opin. Chem. Biol., (2006) 10:598-600.

    Editorial comment on the special biopolymers issue of Curr. Opin. Chem. Biol..

  • Chemical Biology: a broader take on DNA

    A.M. Leconte, F.E Romesberg, Nature, (2006) 444:553-555.

    We review the report of the structure of xDNA from the Kool lab.

  • Direct and high resolution characterization of cytochrome c equilibrium folding

    L.B. Sagle, J. Zimmermann, P.E. Dawson, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2006) 128:14232-14233.

    We report the use of a recently developed technique to study the folding of cyt c that is based on the site-selective incorporation of carbon−deuterium (C−D) bonds and their characterization by IR spectroscopy.

  • Stability and polymerase recognition of pyridine nucleobase analogues: role of minor-groove H-bond acceptors

    Y. Kim, A.M. Leconte, Y. Hari, F.E.Romesberg, Angew. Chem. Int. Ed. (2006) 45:7809-7812.

    Pyridyl nucleoside analogues are synthesized and characterized (see structures). An α-glycosidic nitrogen atom provides an H-bond acceptor that does not significantly facilitate pairing with natural nucleobases. However, it forms minor-groove H-bonds with water molecules and DNA polymerases that optimize the stability and replication, respectively, of the unnatural base pair.

  • Infrared line shape of an alpha-carbon deuterium-labeled amino acid

    C.S. Kinnaman, M.E. Cremeens, F.E. Romesberg, S.A. Corcelli, J. Am. Chem. Soc. (Comm.) (2006) 128(41):13334-13335.

    We explore the viability of α-carbon deuterated bonds (Cα−D) as infrared (IR) probes of protein backbone dynamics.

  • Amplify this! DNA and RNA get a third base pair

    A.M. Leconte and F.E. Romesberg, Nat. Meth. (2006) 3:667-668.

    We review a report by Hirao et al. demonstrating that DNA containing an unnatural base pair is both efficiently PCR amplified and transcribed.

  • Antibody evolution constrains conformational heterogeneity by tailoring protein dynamics

    J. Zimmermann, E.L. Oakman, I.F. Thorpe, X. Shi, P. Abbyad, C.L. Brooks, III, S.G. Boxer, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2006) 103:13722-13727.

    We report the characterization of protein heterogeneity and dynamics as a function of evolution for the antifluorescein antibody 4-4-20. Using nonlinear laser spectroscopy, surface plasmon resonance, and molecular dynamics simulations, we demonstrate that evolution localized the Ab-combining site from a heterogeneous ensemble of conformations to a single conformation by introducing mutations that act cooperatively and over significant distances to rigidify the protein.

  • Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin

    R.T. Cirz, B.M. O'Neill, J.A. Hammond, S.R. Head, F.E. Romesberg, J. Bacteriol. (2006) 188:7101-7110.

    We use DNA microarrays to characterize the global transcriptional response of P. aeruginosa to clinical-like doses of the antibiotic ciprofloxacin and also to determine the component that is regulated by LexA cleavage and the SOS response.[Download Supporting Data][Download SI Methods & Tables]

  • Side effects may include evolution

    R.T. Cirz, N. Gingles, F.R. Romesberg, Nat. Med. (2006) 12:890-891.

    We review a report by Prudhomme et al. in Science that demonstrates how Streptococcus pneumoniae may increase their chances of acquiring exogenous DNA through the upregulation of competence in response to antibiotic treatment.

  • Efforts towards expansion of the genetic alphabet: pyridone and methyl pyridone nucleobases

    A.M. Leconte, S. Matsuda, G.T. Hwang, F.E. Romesberg. Angew. Chem. Int. Ed. (2006) 45:4326-4329.

    Here, we report the synthesis and characterization of nucleotides bearing pyridone nucleobase analogues, which are expected to position their carbonyl group into the minor groove.

  • Redox-coupled dynamics and folding in cytochrome c

    L.B. Sagle, J. Zimmermann, S. Matsuda, P.E. Dawson, F.E. Romesberg, J. Am. Chem. Soc. (2006) 128:7909-7915.

    We incorporated carbon−deuterium bonds throughout cyt c and characterized their absorption frequencies and line widths using IR spectroscopy. Our data suggest that while the oxidized protein is not more flexible than the reduced protein, it is more locally unfolded.

  • The role of Doa1 in the S. cerevisiae DNA damage response

    E.T. Lis, F.E. Romesberg, Mol. Cell. Biol. (2006) 26:4122-4133.

    Here, we describe SS. cerevisiae Doa1, which helps to control the damage response by channeling ubiquitin from the proteosomal degradation pathway into pathways that mediate altered DNA replication and chromatin modification. Our data suggest that Doa1 is the major source of ubiquitin for the DNA damage response and that Doa1 also plays an additional essential and more specific role in the monoubiquitination of histone H2B.

  • An efficiently extended class of unnatural base pairs

    A.M. Leconte, S.Matsuda, F.E.Romesberg, J. Am. Chem. Soc. (Comm.) (2006) 128:6780-6781.

    We describe a simple screen that enables the characterization of large numbers of previously uncharacterized hetero base pairs. From this screen, we identify a class of unnatural base pairs which are extended more efficiently than any unnatural base pair reported to date.

  • Efforts toward developing direct probes of protein dynamics

    M.E. Cremeens, H. Fujisaki, Y. Zhang, J. Zimmermann, L.B. Sagle, S. Matsuda, P.E. Dawson, J.E. Straub, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2006) 128(18):6028-6029.

    We report the first IR characterization of a single C−D bond within a protein, methyl-d1 Met80 of horse heart cytochrome c. A comparison was made to methyl-d1/d3 methionine as well as methyl-d3 Met80.

  • Optimization of unnatural base pair packing for polymerase recognition

    S. Matsuda, A.A. Henry, F.E. Romesberg, J. Am. Chem. Soc. (2006) 128:6369-6375.

    We report the characterization of polymerase-mediated replication of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues.

  • Esc4/Rtt107 and the control of recombination during replication

    J. K. Chin, V. I. Bashkirov, W.-D. Heyer, F.E. Romesberg, DNA Repair (2006) 5:618-628.

    We show that Esc4 interacts with several proteins involved in the repair and processing of stalled or collapsed replication forks, including the recombination protein Rad55, and we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein to recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.

  • Substituent effects on the pairing and polymerase recognition of unnatural base pairs

    G.T. Hwang, F.E. Romesberg, Nucleic Acids Res. (2006) 34:2037-2045.

    We report the synthesis, stability and polymerase recognition of nucleoside analogs bearing single bromo- or cyano-derivatized phenyl rings and find that both modifications generally stabilize base pair formation to a greater extent than methyl or fluoro substitution.

  • Induction and inhibition of ciprofloxacin resistance-conferring mutations in hypermutator bacteria

    R.T. Cirz,F.E. Romesberg, Antimicrob. Agents Chemother. (2006) 50:220-225.

    We show that LexA cleavage and polymerase derepression are required for the evolution of clinically significant resistance in MMR-defective E. coli.

2005

  • Differential solvation and tautomer stability of a model base pair within the minor and major grooves of DNA

    F.-Y. Dupradeau, D.A. Case, C. Yu, R. Jimenez, F.E. Romesberg, J. Am. Chem. Soc. (2005) 127:15612-15617.

    We show that when HBO is positioned in the minor groove of DNA, the enol tautomer of HBO is preferentially stabilized, whereas our previous study showed that when HBO is positioned in the major groove, the keto tautomer is preferentially stabilized. Based on MD simulations, we suggest that this results from the formation of a stable hydrogen-bond between the HBO enol and an H-bond acceptor that is only available in the minor groove.

  • Polymerase evolution: efforts toward expansion of the genetic code

    A.M. Leconte, L.Chen, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2005) 127:12470-12471.

    We report the use of an activity-based selection system to evolve a DNA polymerase that more efficiently replicates DNA containing the PICS self-pair.

  • REVIEW: The evolution of DNA polymerases with novel activities

    A.A. Henry and F.E. Romesberg, Curr. Opin. Biotechnol. (2005) 16:370-377

    We review several activity-based screening and selection approaches that have been used to identify polymerases with novel activities.

  • Inhibition of mutation and combating the evolution of antibiotic resistance

    R.T. Cirz, J.K. Chin, D.R. Andes, V. de Crécy-Lagard, W.A. Craig, F.E. Romesberg, PLoS Biol. (2005) 3:e176.

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  • REVIEW: Directed evolution of novel polymerases

    R.C. Holmberg, A.A. Henry, F.E. Romesberg, Biomol. Eng. (2005) 22:39-49.

    We review screening and selection approaches, both in vivo and in vitro, that have been used to evolve polymerases with a variety of novel properties, such as thermal stability, resistance to inhibitors, and altered substrate specificity.

2004

  • Coordinated functions of WSS1, PSY2, and TOF1 in the DNA damage response

    B.M. O'Neill, D. Hanway, E.A. Winzeler, F.E. Romesberg, Nucleic Acids Res. (2004) 32:6519-6530.

    The stabilization and processing of stalled replication forks is required to maintain genome integrity in all organisms. Based on both physical and genetic interactions detected between WSS1, PSY2, and TOF1, we suggest that Wss1 and Psy2 similarly function to stabilize or process stalled or collapsed replication forks.

  • Optimization of interstrand hydrophobic packing interactions within unnatural base pairs

    S. Matsuda, F.E. Romesberg, J. Am. Chem. Soc. (2004) 126:14419-14427.

    We report the synthesis and stability of unnatural base pairs formed between simple phenyl rings modified at different positions with methyl groups. Several are virtually as stable as a natural base pair in the same sequence context, showing that neither hydrogen-bonding nor large aromatic surface area are required for base pair stability within duplex DNA and that interstrand interactions between small aromatic rings may be optimized for both stability and selectivity.

  • REVIEW: Isotope effects and quantum tunneling in enzyme-catalyzed hydrogen transfer. Part I. The experimental basis

    F.E. Romesberg, R.L. Schowen, Adv. Phys. Org. Chem. (2004) 39:27-77.

    We review the role of quantum tunneling as a mechanism of the enymatic transfer of hydrogenic entities (protons, hydrogen atoms, and hydride ions), including the background of the subject, the conceptual apparatus that underlies the isotopic studies, the phenomenology of the experimental observations, and a qualitative sketch of the interpretative, mechanistic models that have emerged.

  • Efforts to expand the genetic alphabet: identification of a replicable unnatural DNA self-pair

    A.A. Henry, A.G. Olsen, S. Matsuda, C. Yu, B.H. Geierstanger, F.E. Romesberg, J. Am. Chem. Soc. (2004) 126(22):6923-6931.

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  • Preliminary characterization of light harvesting in E. coli DNA photolyase

    A.A. Henry, R. Jimenez, D. Hanway, F.E. Romesberg, Chembiochem (2004) 5:1088-1094.

    E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. Here, we investigate the origins of how the enzyme tunes the photophysical properties of the antenna cofactor appropriately for biological function.

  • A high-resolution probe of protein folding

    L.B. Sagle, J. Zimmermann, P.E. Dawson, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2004) 126:3384-3385.

    To generate residue-specific information on the equilibrium folding of cyt c, we have semisynthesized the protein with specifically deuterated residues. Plotted as a function of added guanidine hydrochloride denaturant, the absorption intensities of the protein C-D bonds reveal that cyt c undergoes a conformational change at the protein-based heme ligand, Met80, which is then followed by a more global unfolding at 2.3 M GdnHCL.

  • Protein dynamics and the immunological evolution of molecular recognition

    R. Jimenez, G. Salazar, J. Yin, T. Joo, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2004) 101:3803-3808.

    3PEPS and steady-state spectroscopy in combination with binding and structural data, are used to examine the immunological evolution of protein flexibility in an anti-fluorescein antibody. We show that antibody dynamics are systematically manipulated during affinity maturation, and suggest that the evolution of protein flexibility may be a central component of the immune response.

  • Expanding the substrate repertoire of a DNA polymerase by directed evolution

    M. Fa, A. Radeghieri, A.A. Henry, F.E. Romesberg, J. Am. Chem. Soc. (2004) 126:1748-1754.

    In this report, mutants that efficiently synthesize an unnatural polymer from 2‘-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of an activity-dependent phage display method. One evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.

2003

2002

  • Enzymatic phosphorylation of unnatural nucleosides

    Y. Wu, M. Fa, E.L. Tae, P.G. Schultz, F.E. Romesberg, J. Am. Chem. Soc.(2002) 124:14626-14630.

    We report our initial efforts toward the development of an unnatural in vivo nucleoside phosphorylation pathway that is based on nucleoside salvage enzymes.

  • Excited state dynamics and heterogeneity of folded and unfolded states of cytochrome c

    R. Jimenez, F.E. Romesberg, J. Phys. Chem. B. (2002) 106(35):9172-9180.

    Transient absorption, transient grating, and 3PEPS measurements are used to characterize the photophysics of the heme chromophore in the folded protein and in two different unfolded proteins. We find that cyt c has little structural heterogeneity in the folded state, and a larger degree of structural heterogeneity in the unfolded states that depends on the unfolding conditions.

  • Polymerase recognition of unnatural base pairs

    C. Yu, A.A. Henry, F.E. Romesberg, P.G. Schultz, Angew. Chem. Int. Ed. Engl. (2002) 41:3841-3844.

    Heteroatom substitution of ICS via replacement of C6 with nitrogen and thio substitution at C10 provides the base SNICS, which forms stable self-pairs and can therefore be used for efficient unnatural base pair replication.

  • REVIEW: Development of a universal nucleobase and modifed nucleobases for expanding the genetic code

    F.E. Romesberg, C. Yu, S. Matsuda, A.A. Henry, Curr. Prot. Nucleic Acid Chem. (2002) Unit 1.5.

    We describe methods for the synthesis and characterization of modified nucleobases.

  • Previously uncharacterized genes in the UV- and MMS-induced DNA damage reponse in yeast

    D. Hanway, J.K. Chin, G. Xia, G. Oshiro, E.A. Winzeler, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2002) 99:10605-10610.

    A competitive growth assay is used to identify yeast genes involved in the repair of UV- or MMS-induced DNA damage using a collection of 2,827 yeast strains in which each strain has a single ORF replaced with a cassette containing two unique sequence tags. Identified genes include three uncharacterized ORFs, as well as genes that encode protein products implicated in ubiquitination, gene silencing, and transport across the mitochondrial membrane.

  • Directed evolution of novel polymerase activities: mutation of a DNA polymerase into and efficient RNA polymerase

    G. Xia, L. Chen, T. Sera, M. Fa, P.G. Schultz, F.E. Romesberg, Proc. Natl. Acad. Sci. USA (2002) 99:6597-6602.

    We use an activity-based phage display method to screen a Stoffel fragment (SF) polymerase library, and we identify three SF mutants that incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type SF polymerase incorporates dNTP substrates.

  • Solvent-Dependent Photoinduced Tautomerization of 2-(2'-Hydroxyphenyl)benzoxazole

    O.K. Abou-Zied, R. Jimenez, E.H.Z. Thompson, D.P. Millar, F.E. Romesberg, J. Phys. Chem. A. (2002) 106:3665-3672.

    The solvent-dependent ground-state conformational equilibrium and excited-state dynamics of 2-(2‘-hydroxyphenyl)benzoxazole are characterized in several solvents on the femtosecond to nanosecond time scales.

  • Protein dynamics and cytochrome c: correlations between ligand vibrations and redox activity, J. Am. Chem. Soc. (Comm.) (2002) 124:1846-1847.

    J.K. Chin, R. Jimenez, F.E. Romesberg,

    The dynamics of the protein-based methionine heme ligand were examined by selectively deuterating the ligand's methyl group. The frequency and line width of the C−D bonds were easily observable and shown to be sensitive to mutation-induced changes in the protein redox potential.

  • Flexibility of an Antibody Binding Site Measured with Photon Echo Spectroscopy

    R. Jimenez, D.A. Case, F.E. Romesberg, J. Phys. Chem. B. (2002) 106(5):1090-1103.

    The reorganization energies of the chromophore−antibody complexes are determined and are found to be distributed over a wide range. 3PEPS spectroscopy is used to measure the time scales of the reorganization process in one complex. We find that the antibody motions occur on time scales ranging from 75 femtoseconds to 67 picoseconds.

  • Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues

    M. Berger, S.D. Luzzi, A.A. Henry, F.E. Romesberg, J. Am. Chem. Soc. (2002) 124:1222-1226.

    Here, we examine thiophene and furan heterocycles as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases.

2001

2000

  • Chemically reactive immunogens lead to functional convergence of the immune response

    H. Shulman, C. Makarov, A.K. Ogawa, F.E. Romesberg, E. Keinan, J. Am. Chem. Soc. (2000) 122:10743-10753.

    Hammett correlation studies of the aldol and retroaldol reactions catalyzed by antibodies 38C2 and 24H6 reveal that although the two antibodies have broad substrate specificities, they utilize slightly different mechanisms. Antibody 38C2 adopts a mechanism that is reminiscent of an acid-catalyzed aldol reaction, while antibody 24H6 follows a mechanism that is similar to the base-catalyzed reaction.

  • A Novel Copper-Mediated DNA Base Pair

    E. Meggers, P.L. Holland, W.B. Tolman, F.E. Romesberg, P.G. Schultz, J. Am. Chem. Soc. (Comm.) (2000) 122:10714-10715.

    We describe a base pair with a pyridine-2,6-dicarboxylate nucleobase (Dipic) as a planar tridentate ligand, and a pyridine nucleobase (Py) as the complementary single donor ligand. Characterization of this base pair in duplex DNA shows that interbase metal coordination can replace the hydrogen bonding schemes found in the natural base pairs dA:dT and dG:dC.

  • A Phototautomerizable Model DNA Base Pair

    A.K. Ogawa, O.K. Abou-Zied, V. Tsui, R. Jimenez, D.A. Case, F.E. Romesberg,

    2-(2‘-Hydroxyphenyl)benzoxazole (HBO) undergoes rapid photoinduced proton transfer from the enol-imine to the keto-amine tautomer. When incorporated in duplex DNA opposite an abasic site, HBO appears to be a good mimic of a natural DNA base pair based on duplex stability, UV and CD spectroscopy, and molecular dynamics simulations.

  • Rational Design of an Unnatural Base Pair with Increased Kinetic Selectivity

    A.K. Ogawa, Y. Wu, M. Berger, P.G. Schultz, F.E. Romesberg, J. Am. Chem. Soc. (Comm.) (2000) 122:8803-8804.

    Here we report the synthesis and characterization of 3MN and 2Np, and we show that the 3MN:3MN self-pair is synthesized by KF with an efficiency of 2.4 × 106 M-1min-1.

  • Stable and selective hybridization of oligonucleotides with unnatural hydrophobic bases

    M. Berger, A.K. Ogawa, D.L. McMinn, Y. Wu, P.G. Schultz, F.E. Romesberg, Angew. Chem. Int. Ed. Engl. (2000) 39:2940-2942.

    Relying on only interbase hydrophobic packing for bonding, several unnatural nucleobases are reported that form selective and stable self-pairs in duplex DNA. These unnatural nucleobases should make it possible to increase the stringency and information content of hybridization or encoding experiments.

  • Efforts toward expansion of the genetic alphabet: optimization of interbase hydrophobic interactions

    Y. Wu, A.K. Ogawa, M. Berger, D.L. McMinn, P.G. Schultz, F.E. Romesberg, J. Am. Chem. Soc. (2000) 122(32):7621-7632

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  • Efforts toward expansion of the genetic alphabet: information storage and replication with unnatural hydrophobic base pairs

    A.K. Ogawa, Y. Wu, D.L. McMinn, J. Liu, P.G. Schultz, F.E. Romesberg, J. Am. Chem. Soc. (2000) 122(14):3274-3287.

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  • Universal bases for hybridization, replication and chain termination

    M. Berger, Y. Wu, A.K. Ogawa, D.L. McMinn, P.G. Schultz, F.E. Romesberg, Nucleic Acids Res. (2000) 28:2911-2914.

    The reported unnatural, predominantly hydrophobic nucleoside analogs are efficient ‘universal bases’ for hybridization, template directed DNA synthesis and chain termination. Moreover, several of the universal bases function in their biological role, hybridization or replication, with an efficiency not significantly reduced relative to their natural counterparts

1999