The 518 protein kinases encoded in the human genome are exquisitely regulated and their aberrant function(s) are often associated with human disease. Thus, in order to advance therapeutics and to probe signal transduction cascades there is considerable interest in the development of inhibitors that can selectively target protein kinases. However, identifying specific compounds against such a large array of protein kinases is difficult to routinely achieve utilizing traditional activity assays, where purified protein kinases are necessary. Toward a simple, rapid, and practical method for identifying specific inhibitors, we describe the development and application of a split-protein methodology utilizing a coiled-coil assisted three-hybrid system. In this approach, a protein kinase of interest is attached to the C-terminal fragment of split-firefly luciferase and the coiled-coil Fos, which is specific for the coiled-coil Jun, is attached to the N-terminal fragment. Upon addition of Jun conjugated to a pan-kinase inhibitor such as staurosporine, a three-hybrid complex is established with concomitant reassembly of the split-luciferase enzyme. An inhibitor can be potentially identified by the commensurate loss in split-luciferase activity by displacement of the modified staurosporine. We demonstrate that this new three-hybrid approach is potentially general by testing protein kinases from the different kinase families. To interrogate whether this method allows for screening inhibitors, we tested six different protein kinases against a library of 80 known protein kinase inhibitors. Finally, we demonstrate that this three-hybrid system can potentially provide a rapid method for structure/function analysis as well as aid in the identification of allosteric inhibitors.

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Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.

We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.

Proteases are widely studied as they are integral players in cell cycle control and apoptosis. We report a new approach for the design of a family of genetically encoded turn-on protease biosensors. In our design, an auto-inhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new auto-inhibition design paradigm, we present the rational construction and optimization of three generations of protease biosensors, with the final design providing a 1000 fold increase in bioluminescent signal upon addition of the TEV protease. We demonstrate the generality of the approach utilizing two different split-protein reporters, firefly luciferase and beta-lactamase, while also testing our design in the context of a therapeutically relevant protease, caspase-3. Finally, we present a dual-protease sensor geometry that allows for the use of these turn-on sensors as potential AND logic gates. Thus these studies potentially provide a new method for the design and implementation of genetically encoded turn-on protease sensors while also providing a general auto-inhibited coiled-coil strategy for controlling the activity of fragmented proteins.

The methylation pattern of genes at CpG dinucleotide sites is an emerging area in epigenetics. Furthermore, the hypermethylation profiles of tumor suppressor genes are linked to specific tumor types. Thus, new molecular approaches for the rapid determination of the methylation status of these genes could provide a powerful method for early cancer diagnosis as well as insight into mechanisms of epigenetic regulation of genetic information. Toward this end, we have recently reported the first design of a split-protein sensor for the site-specific detection of DNA methylation. In this approach a split green fluorescent protein reporter provided a sequence-specific readout of CpG methylation. In the present work, we describe a sensitive second-generation methylation detection system that utilizes the split enzymatic reporter, TEM-1 beta-lactamase, attached to specific DNA binding elements. This system, termed mCpG-SEER-beta-Lac, shows a greater than 40-fold specificity for methylated versus nonmethylated CpG target sites. Importantly, the resulting signal enhancement afforded by the catalytic activity of split-beta-lactamase allowed for the sensitive detection of 2.5 fmol of methylated target dsDNA in 5 min. Thus, this new sensor geometry represents a 250-fold enhancement in assay time and a 2000-fold enhancement in sensitivity over our first-generation system for the detection of specific sites of DNA methylation.

IntroductionThe ability to conditionally elicit a response in the presence of a user-defined macromolecular target has great potential in biosensor design, targeted therapeutics, and a variety of applications in synthetic biology. With our enhanced understanding of the human genome, both DNA and RNA have become increasingly important biological targets related to diverse human disease. 1 General methods for nucleic acid targeting now include several hybridization based approaches such as RNA interference (RNAi), triplex-forming oligonucleotides (TFOs), peptide nucleic acids (PNAs), as well as the elegantly designed polyamides. 2 Nucleic acid detection most commonly relies on fluorescently labeled oligonucleotides, as in the case of fluorescence in situ hybridization (FISH). 3 However, the constitutive fluorescent signal associated with this class of probes necessitates washing steps and results in reduced sensitivity, which potentially limits the utility of this technique for in vivo imaging. 4 To address these concerns, recent efforts have been directed toward generating "turn-on" fluorescent or electrochemical sensors, which couple conditional signal output to target binding. 5 In a complementary approach, there has been recent interest in DNA-and RNA-templated reactions, wherein probe localization on a single-stranded nucleic acid target enables the specific chemical transformation of attached moieties. 6 Examples of nucleic acid-templated chemical reactions include FRET-and quencher-based autoligation probes, metallosalen−DNA conjugates and deoxyribozymes for DNA hydrolysis, and catalytically released cytotoxic drugs. 7 Additionally, there is great interest in the design of DNA-directed gain of function proteins, which cannot only serve as sensors and genome modifying agents, but also have potential as specific therapeutics. Many groups have described protein-based approaches for the conditional reassembly of fragmented proteins on double-stranded DNA (dsDNA) mainly utilizing the Cys 2 His 2 class of zinc fingers (ZFs) as targeting domains. 8, 9 Published in Journal of the American Chemical Society 132:33 (2010) Corresponding author -I. Ghosh, ghosh@email.arizona.edu AbstractThe ability to conditionally turn on a signal or induce a function in the presence of a user-defined RNA target has potential applications in medicine and synthetic biology. Although sequence-specific pumilio repeat proteins can target a limited set of ss-RNA sequences, there are no general methods for targeting ssRNA with designed proteins. As a first step toward RNA recognition, we utilized the RNA binding domain of argonaute, implicated in RNA interference, for specifically targeting generic 2-nucleotide, 3′ overhangs of any dsRNA. We tested the reassembly of a split-luciferase enzyme guided by argonaute-mediated recognition of newly generated nucleotide overhangs when ssRNA is targeted by a designed complementary guide sequence. This approach was successful when argonaute was utilized in conjunction with a pumilio repeat and expa...

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