Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a non-specific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone non-homologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of CRISPR/Cas-based RNA-guided DNA endonucleases.

Most enzymatic transformations have a synthetic counterpart. Often though, the mechanisms by which natural and synthetic catalysts operate differ markedly. The catalytic asymmetric aldol reaction as a fundamental C-C bond forming reaction in chemistry and biology is an interesting case in this respect. Chemically, this reaction is dominated by approaches that utilize preformed enolate equivalents in combination with a chiral catalyst. 1 Typically, a metal is involved in the reaction mechanism. 1d Most enzymes, however, use a fundamentally different strategy and catalyze the direct aldolization of two unmodified carbonyl compounds. Class I aldolases utilize an enamine based mechanism, 2 while Class II aldolases mediate this process by using a zinc cofactor. 3 The development of aldolase antibodies that use an enamine mechanism and accept hydrophobic organic substrates has demonstrated the potential inherent in amine-catalyzed asymmetric aldol reactions. 4 Recently, the first small-molecule asymmetric class II aldolase mimics have been described in the form of zinc, lanthanum, and barium complexes. 5,6 However, amine-based asymmetric class I aldolase mimics have not been described in the literature. 7 Here we report our finding that the amino acid proline is an effective asymmetric catalyst for the direct aldol reaction between unmodified acetone and a variety of aldehydes.Recently we developed broad scope aldolase antibodies that show very high enantioselectivities, have enzymatic rate accelerations, and use the enamine mechanism of class I aldolases. 4 During the course of these studies, we found that one of our aldolase catalytic antibodies (Aldolase Antibody 38C2, Aldrich) is an efficient catalyst for enantiogroup-differentiating aldol cyclodehydrations of 2,6-heptanediones to give cyclohexenones, including the Wieland-Miescher ketone. 8,9 These intramolecular reactions are also catalyzed by proline (Hajos-Eder-Sauer-Wiechert reaction) 10 and it has been postulated that they proceed via an enamine mechanism. 11 However, the proline-catalyzed direct intermolecular asymmetric aldol reaction has not been described. Further, there are no asymmetric small-molecule aldol catalysts that use an enamine mechanism. 7 Based on our own results and Shibasaki's work on lanthanum-based small-molecule aldol catalysts, 4,6 we realized the great potential of catalysts for the direct asymmetric aldol reaction.We initially studied the reaction of acetone with 4-nitrobenzaldehyde. Reacting proline (30 mol %) in DMSO/acetone (4:1) with 4-nitrobenzaldehyde at room temperature for 4 h furnished aldol product (R)-1 in 68% yield and 76% ee (eq 1). This result is quite remarkable since it is known that proline can undergo a variety of reactions with aldehydes. For example, aliphatic aldehydes react with proline to give either the oxazolidinone and/ or various other compounds, including products of self-aldolization. 12 Aromatic aldehydes (including 4-nitrobenzaldehyde) can condense with proline to form azomethine ylides that undergo ...

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.

The ability of antibodies to neutralize diverse primary isolates of human immunodeficiency virus-type 1 in vitro has been questioned, with implications for the likely efficacy of vaccines. A recombinant human antibody to envelope glycoprotein gp120 was generated and used to show that primary isolates are not refractory to antibody neutralization. The recombinant antibody neutralized more than 75 percent of the primary isolates tested at concentrations that could be achieved by passive immunization, for example, to interrupt maternal-fetal transmission of virus. The broad specificity and efficacy of the antibody implies the conservation of a structural feature on gp120, which could be important in vaccine design.

ErbB2 is a receptor tyrosine kinase whose activity in normal cells depends on dimerization with another ligand-binding ErbB receptor. In contrast, amplification of c-erbB2 in tumors results in dramatic overexpression and constitutive activation of the receptor. Breast cancer cells overexpressing ErbB2 depend on its activity for proliferation, because treatment of these cells with ErbB2-specific antagonistic antibodies or kinase inhibitors blocks tumor cells in the G 1 phase of the cell cycle. Intriguingly, loss of ErbB2 signaling is accompanied by a decrease in the phosphotyrosine content of ErbB3. On the basis of these results, it has been proposed that ErbB3 might be a partner for ErbB2 in promoting cellular transformation. To test this hypothesis and directly examine the role of the ''kinase dead'' ErbB3, we specifically ablated its expression with a designer transcription factor (E3). By infection of ErbB2-overexpressing breast cancer cells with a retrovirus expressing E3, we show that ErbB3 is an essential partner in the transformation process. Loss of functional ErbB2 or ErbB3 has similar effects on cell proliferation and cell cycle regulators. Furthermore, expression of constitutively active protein kinase B rescues the proliferative block induced as a consequence of loss of ErbB2 or ErbB3 signaling. These results demonstrate that ErbB2 overexpression and activity alone are insufficient to promote breast tumor cell division. Furthermore, we identify ErbB3's role, which is to couple active ErbB2 to the phosphatidylinositol 3-kinase͞protein kinase B pathway. Thus, the ErbB2͞ErbB3 dimer functions as an oncogenic unit to drive breast tumor cell proliferation.T he family of ErbB receptor tyrosine kinases includes four members: epidermal growth factor (EGF) receptor͞ErbB1, ErbB2, ErbB3, and ErbB4. Binding of peptides of the EGFrelated growth factor family to the extracellular domain of ErbB receptors results in the formation of homo-and heterodimers. Ligand binding induces the intrinsic receptor kinase activity, ultimately leading to stimulation of intracellular signaling cascades (1, 2). The physiological role of ErbB2, in the context of ErbB ligand signaling, is to serve as a coreceptor (3, 4). In fact, ErbB2 appears to be the preferred partner of the other ligandbound ErbBs (5, 6). The importance of heterodimer-mediated signaling in normal development is obvious from studies in genetically modified mice. This is particularly true for ErbB2͞ ErbB3 and ErbB2͞ErbB4 heterodimers. Loss of ErbB2 or ErbB3 has a similar impact on neuronal development (7), whereas loss of ErbB2 or ErbB4 has major effects on heart development (8, 9).A wealth of clinical data has demonstrated that ErbB receptor tyrosine kinases, in particular ErbB1 and ErbB2, have roles in human cancer development, thus making them attractive targets for cancer therapies (10-13). ErbB2 overexpression, generally attributable to gene amplification, occurs in 25-30% of breast cancer and correlates with shorter time to relapse and lower overall survival (1...

A phagemid system was developed for the monovalent display of combinatorial antibody Fab libraries on the surface of filamentous phage M13. Fab fragments were fused to the carboxyl-terminal domain of the gene HI protein.Phage displaying Fab fragments on their surface, or Phabs, were enriched by 103-to 105-fold on antigen-coated surfaces over nonspecific phage. The method may replace current antibody cloning techniques.Our combinatorial approach (1,2) provides a means of capturing the vast diversity of the immunological repertoire (1-5). The approach relies on the ability to clone antibody heavy-and light-chain fragments independently and randomly recombine them in a system that allows the specificity of binding to be probed. Previously, we used a A phage system that allowed probing of plaque-lifts for the identification of desired clones. This system is limited by the size of the combinatorial library that may be examined (-106 members).An interesting approach to accessing larger libraries involves their expression on the surface of filamentous phage. The display of libraries of small peptides on the surface of filamentous phage has proven to be a powerful approach for selecting ligands of defined specificity (6-8). Phage display libraries are rapidly being extended to whole proteins. Two monomeric proteins, a single-chain antibody (9) and human growth hormone (10), have been successfully expressed as fusions with the gene III (gIII) product coat protein III (cpIII). We have recently reported the expression and assembly of considerably larger and more complex (-50-kDa heterodimers) proteins, antibody Fab fragments, as fusions with the gene VIII (gVIII) product coat protein VIII (cpVIII) (11).The ability to rapidly sort large combinatorial Fab libraries is particularly important for the development of catalytic antibodies (12). Here we report a strategy based on gIII fusions, complementing our gVIII approach, for the construction, selection, and production of high-affinity antigenspecific Fabs from combinatorial antibody libraries. The combinatorial antibody technology reported here may replace current hybridoma methods for the isolation of monoclonal antibodies.MATERIALS AND METHODS Vector Construction. The pelB leader sequences and cloning sites for the heavy-chain fragment and light chain were derived from phagemids excised from A Hc2 and A Lc2 A vectors as described (2). The sequences were modified to remove a redundant Sac I site from Hc2 phagemid and a Spe I site from the Lc2 phagemid. The combinatorial phagemid vector pComb was constructed from these two modified phagemids by restricting each with Sca I and EcoRI and combining them in a ligation reaction. Recombinants were screened for the presence of two Not I sites yielding the combinatorial vector pComb. The tether sequence GGGGS and gIIl fragment from Spe I to Nhe I were the product of PCR of M13mpl8 (13) using the oligonucleotides 5'-GAGACGACTAGTGGTGGCGGTGGCTCTCCAT-TCGTTTGTGAATATCAA-3' and 5'-TTACTAGCTAG-CATAATAACGGAATACCCAAAAGAACTGG-3'.The la...

Direct asymmetric catalytic aldol reactions have been successfully performed using aldehydes and unmodified ketones together with commercially available chiral cyclic secondary amines as catalysts. Structure-based catalyst screening identified L-proline and 5,5-dimethyl thiazolidinium-4-carboxylate (DMTC) as the most powerful amino acid catalysts for the reaction of both acyclic and cyclic ketones as aldol donors with aromatic and aliphatic aldehydes to afford the corresponding aldol products with high regio-, diastereo-, and enantioselectivities. Reactions employing hydroxyacetone as an aldol donor provide anti-1,2-diols as the major product with ee values up to >99%. The reactions are assumed to proceed via a metal-free Zimmerman-Traxler-type transition state and involve an enamine intermediate. The observed stereochemistry of the products is in accordance with the proposed transition state. Further supporting evidence is provided by the lack of nonlinear effects. The reactions tolerate a small amount of water (<4 vol %), do not require inert reaction conditions and preformed enolate equivalents, and can be conveniently performed at room temperature in various solvents. In addition, reaction conditions that facilitate catalyst recovery as well as immobilization are described. Finally, mechanistically related addition reactions such as ketone additions to imines (Mannich-type reactions) and to nitro-olefins and alpha,beta-unsaturated diesters (Michael-type reactions) have also been developed.

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5-GNN-3 subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9-or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5-(GNN) 6 -3 family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5-untranslated region of this gene was converted into a transcriptional repressor by fusion with Krüppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that genespecific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.