Evaluating the global CpG methylation status of native DNA utilizing a bipartite split-luciferase sensor.

Epigenetic modifications play an essential role in the regulation of gene expression and ultimately cell fate. Methylation of cytosine at CpG dinucleotides (mCpG) is an important epigenetic mark that has been correlated with cancer when present at promoter sites of tumor suppressor genes. To develop a rapid methodology for the direct assessment of global levels of DNA methylation, we first interrogated the methyl-CpG binding domains (MBDs), the Kaiso family of Cys(2)-His(2) zinc fingers, and an SET- and RING-associated domain using a split-luciferase reassembly methodology. We identified MBD1 as the most selective domain for the discrimination between mCpG and CpG sites with over 90-fold selectivity. Utilizing a bipartite strategy, we constructed a purely methylation-dependent bipartite sensor for the direct detection of global levels of DNA methylation by attaching MBD1 domains to each of the split-luciferase halves. This new sensor was validated for the direct determination of genomic DNA methylation levels in in vitro studies without any intervening chemical or enzymatic processing of DNA. Finally, we demonstrated that this bipartite sensor can be utilized for monitoring dose-dependent changes in global levels of methylation in DNA from HeLa cells challenged with 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor.

Profiling small molecule inhibitors against helix-receptor interactions: the Bcl-2 family inhibitor BH3I-1 potently inhibits p53/hDM2.

We validate a practical methodology for the rapid profiling of small molecule inhibitors of protein-protein interactions. We find that a well known BH3 family inhibitor can potently inhibit the p53/hDM2 interaction.

Seeing genetic and epigenetic information without DNA denaturation using sequence-enabled reassembly (SEER).

Virtually all methods for reading the sequence of bases in DNA rely on the ability to denature double-stranded DNA into single strands and then use Watson-Crick base-pairing rules to hybridize the strands with high specificity to another DNA primer or probe. However, nature frequently uses an alternative method, reading the sequence information directly from double-stranded DNA using sequence-specific DNA-binding proteins. Here we describe methods for the construction and testing of sequence probes based on engineered zinc finger DNA-binding proteins. Background is reduced using split-reporter molecules, and signal is amplified using enzymatic reporters. The resulting sequence-enabled reassembly (SEER) probes can be configured to detect DNA sequence (genetic) or DNA methylation (epigenetic) information.

A general approach for receptor and antibody-targeted detection of native proteins utilizing split-luciferase reassembly.

The direct detection of native proteins in heterogeneous solutions remains a challenging problem. Standard methodologies rely on a separation step to circumvent nonspecific signal generation. We hypothesized that a simple and general method for the detection of native proteins in solution could be achieved through ternary complexation, where the conditional signal generation afforded by split-protein reporters could be married to the specificity afforded by either native receptors or specific antibodies. Toward this goal, we describe a solution phase split-luciferase assay for native protein detection, where we fused fragmented halves of firefly luciferase to separate receptor fragments or single-chain antibodies, allowing for conditional luciferase complementation in the presence of several biologically significant protein targets. To demonstrate the utility of this strategy, we have developed and validated assay platforms for the vascular endothelial growth factor, the gp120 coat protein from HIV-1, and the human epidermal growth factor receptor 2 (HER2), a marker for breast cancer. The specificities of the recognition elements, CD4 and the 17b single-chain antibody, employed in the gp120 sensor allowed us to parse gp120s from different clades. Our rationally designed HER2 sensing platform was capable of discriminating between HER2 expression levels in several tumor cell lines. In addition, luminescence from reassembled luciferase was linear across a panel of cell lines with increasing HER2 expression. We envision that the proof of principle studies presented herein may allow for the potential detection of a broad range of biological analytes utilizing ternary split-protein systems.

Toward a general approach for RNA-templated hierarchical assembly of split-proteins.

The 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 ssRNA 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 expanded the scope of potential ssRNA targets. However, targeting any desired ssRNA remained elusive as two argonaute domains provided minimal reassembled split-luciferase. We next designed and tested a second hierarchical assembly, wherein ssDNA guides are appended to DNA hairpins that serve as a scaffold for high affinity zinc fingers attached to split-luciferase. In the presence of a ssRNA target containing adjacent sequences complementary to the guides, the hairpins are brought into proximity, allowing for zinc finger binding and concomitant reassembly of the fragmented luciferase. The scope of this new approach was validated by specifically targeting RNA encoding VEGF, hDM2, and HER2. These approaches provide potentially general design paradigms for the conditional reassembly of fragmented proteins in the presence of any desired ssRNA target.

A coiled-coil enabled split-luciferase three-hybrid system: applied toward profiling inhibitors of protein kinases.

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.

An autoinhibited coiled-coil design strategy for split-protein protease sensors.

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 autoinhibited coiled-coil switch is turned on upon proteolytic cleavage, which results in the complementation of split-protein reporters. Utilizing this new autoinhibition 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 autoinhibited coiled-coil strategy for controlling the activity of fragmented proteins.

A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters.

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.

Split beta-lactamase sensor for the sequence-specific detection of DNA methylation.

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.

DNA sequence-enabled reassembly of the green fluorescent protein.

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.