Chemistry Commons^™

A Turn-On Split-Luciferase Sensor For The Direct Detection Of Poly(Adp-Ribose) As A Marker For Dna Repair And Cell Death, Jennifer L. Furman, Pui-Wing Mok, Shengyi Shen, Cliff I. Stains, Indraneel Ghosh

Cliff Stains Publications

Designed sensors comprising split-firefly luciferase conjugated to tandem poly(ADP-ribose) binding domains allow for the direct solution phase detection of picogram quantities of PAR and for monitoring temporal changes in poly(ADP-ribosyl)ation events in mammalian cells.

Includes Supplementary Material.

A P38Α-Selective Chemosensor For Use In Unfractionated Cell Lysates, Cliff I. Stains, Elvedin Luković, Barbara Imperiali

Cliff Stains Publications

Recent efforts have identified the p38α Ser/Thr kinase as a potential target for the treatment of inflammatory diseases as well as non-small cell lung carcinoma. Despite the significance of p38α, no direct activity probe compatible with cell lysate analysis exists. Instead, proxies for kinase activation, such as phosphospecific antibodies, which do not distinguish between p38 isoforms, are often used. Our laboratory has recently developed a sulfonamido-oxine (Sox) fluorophore that undergoes a significant increase in fluorescence in response to phosphorylation at a proximal residue, allowing for real-time activity measurements. Herein we report the rational design of a p38α-selective chemosensor using this …

A General Approach For Receptor And Antibody-Targeted Detection Of Native Proteins Utilizing Split-Luciferase Reassembly, Cliff I. Stains, Jennifer L. Furman, Jason R. Porter, Srivats Rajagopal, Richard T. Wyatt, Indraneel Ghosh

Cliff Stains Publications

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 …

Toward A General Approach For Rna-Templated Hierarchical Assembly Of Split-Proteins, Jennifer L. Furman, Ahmed H. Badran, Oluyomi Ajulo, Jason R. Porter, Cliff I. Stains, David J. Segal, Indraneel Ghosh

Cliff Stains Publications

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 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 …

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, Jason R. Porter, Cliff I. Stains, Benjamin W. Jester, Indraneel Ghosh

Cliff Stains Publications

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, β-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 …

Molecules That Target Beta-Amyloid, Cliff I. Stains, Kalyani Mondal, Indraneel Ghosh

Cliff Stains Publications

The devastating effects of Alzheimer’s and related amyloidogenic diseases have inspired the synthesis and evaluation of numerous ligands to understand the molecular mechanism of the aggregation of the beta-amyloid peptide. Our review focuses on the current knowledge in this field with respect to molecules that have been demonstrated to interact with either oligomeric or fibrillar forms of the beta-amyloid peptide. We describe natural proteins, peptides, peptidomimetics, and small molecules that have been found to interfere with beta-amyloid aggregation. We also detail recent efforts in selecting molecules that target beta-amyloid isolated from antibody, protein, and peptide libraries. These new molecules will …

Direct Detection Of Double-Stranded Dna: Molecular Methods And Applications For Dna Diagnostics, Indraneel Ghosh, Cliff I. Stains, Aik T. Ooi, David J. Segal

Cliff Stains Publications

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving …

Sequence-Enabled Reassembly (Seer) Peptides For The Detection Of Dna Sequences, Aik T. Ooi, Cliff I. Stains, Jason R. Porter, Indraneel Ghosh, David J. Segal

Cliff Stains Publications

By combining custom zinc finger (ZF) DNA-binding technology [1,2] with protein fragment complementation [3], we have developed a technology, designated SEER (Sequence-Enabled Reassembly), that has the potential to “see” or detect genetic information within a living cell (Figure 1). These agents consist of two inactive parts of signal-generating peptides that have the ability to recognize specific DNA sequences. The two parts bind near each other in the presence of a user-defined DNA target site and generate a fluorescent signal. Two prototype SEER systems have been constructed, based on the reassembly of green fluorescent protein (SEERGFP) [4] and the enzyme β-lactamase …