Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and Western blot are common bioanalytical techniques. Successful execution traditionally requires the use of one or more commercially available antibody–small-molecule dyes or antibody–reporter protein conjugates that recognize relatively short peptide tags (<15 amino acids). However, the size of antibodies and their molecular complexity (by virtue of post-translational disulfide formation and glycosylation) typically require either expression in mammalian cells or purification from immunized mammals. The preparation and purification of chemical dye– or reporter protein–antibody conjugates is often complicated and expensive and not commonplace in academic laboratories. In response, researchers have developed comparatively simpler protein scaffolds for macromolecular recognition, which can be expressed with relative ease in E. coli and can be evolved to bind virtually any target. Nanobodies, a minimalist scaffold generated from camelid-derived heavy-chain IgGs, are one such example. A multitude of nanobodies have been evolved to recognize a diverse array of targets, including a short peptide. Here, this peptide tag (termed BC2T) and BC2 nanobody–dye conjugates or reporter protein fusions are evaluated in ELISA, flow cytometry, and Western blot experiments and compared to analogous experiments using commercially available antibody–conjugate/peptide tag pairs. Collectively, the utility and practicality of nanobody-based reagents in bioanalytical chemistry is demonstrated.

Currently, 7 of the top 10 selling drugs are biologics, and all of them are proteins. Their large size, structural complexity, and molecular diversity often results in surfaces capable of potent and selective recognition of receptors that challenge, or evade, traditional small molecules. However, most proteins do not penetrate the lipid bilayer exterior of mammalian cells. This severe limitation dramatically limits the number of disease-relevant receptors that proteins can target and modulate. Given the major role proteins play in modern medicine, and the magnitude of this limitation, it is unsurprising that an enormous amount of effort has been dedicated to overcoming this pesky impediment. In this article, we summarize and evaluate current approaches for intracellular delivery of exogenous proteins to mammalian cells and, in doing so, aim to illuminate fertile ground for future discovery in this critical area of research.

Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5′-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents–small molecule, nucleic acid, or protein–reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of disease-relevant RNA.

Overexpression of the ankyrin repeat oncoprotein gankyrin is directly linked to the onset, proliferation, and/or metastasis of many cancers. The role of gankyrin in multiple disease-relevant biochemical processes is profound. In addition to other cellular processes, gankyrin overexpression leads to decreased cellular levels of p53, through a complex that involves MDM2. Thus, inhibition of this interaction is an attractive strategy for modulating oncogenic phenotypes in gankyrin-overexpressing cells. However, the lack of well-defined, hydrophobic, small-molecule binding pockets on the putative ankyrin repeat binding face presents a challenge to traditional small-molecule drug discovery. In contrast, by virtue of their size and relatively high folding energies, synthetic gankyrin-binding proteins could, in principle, compete with physiologically relevant PPIs involving gankyrin. Previously, we showed that a shape-complementary protein scaffold can be resurfaced to bind gankyrin with moderate affinity (KD ∼6 μM). Here, we used yeast display high-throughput screening, error-prone PCR, DNA shuffling, and protein engineering to optimize this complex. The best of these proteins bind gankyrin with excellent affinity (KD ∼21 nM), selectively co-purifies with gankyrin from a complex cellular milieu, modulates an interaction between gankyrin and a physiological binding partner (S6 ATPase), and suppresses gankyrin/MDM2-dependent ubiquitination of p53.

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

A multitude of RNA hairpins are directly implicated in human disease. Many of these RNAs are potentially valuable targets for drug discovery and basic research. However, very little is known about the molecular requirements for achieving sequence-selective recognition of a particular RNA sequence and structure. Although a relatively modest number of synthetic small to medium-sized RNA-binding molecules have been reported, rapid identification of sequence-selective RNA-binding molecules remains a daunting challenge. RNA recognition motif (RRM) domains may represent unique privileged scaffolds for the generation of synthetic proteins that selectively recognize structured disease-relevant RNAs, including RNA hairpins. As a demonstration of this potential, we mutated putative RNA-binding regions within the U1A RRM and a variant thereof and screened these synthetic proteins for affinity to HIV-1 trans-activation response (TAR) element hairpin RNA. Some of these U1A-derived proteins bind TAR with single-digit micromolar dissociation constants, and they do so preferentially over the native protein’s original target RNA (U1hpII) and a DNA TAR variant. Binding affinity is not appreciably diminished by addition of 10 molar equivalents of cellular tRNAs from Escherichia coli. Taken together, our findings represent the first synthetic RRMs that selectively bind a disease-relevant RNA hairpin and may represent a general approach for achieving sequence-selective recognition of RNA hairpins, which are the focus of therapeutic discovery and basic research.

Increased cellular levels of protein–protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein–protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin’s putative binding face and large surface areas involved in gankyrin-dependent protein–protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5 × 109 library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

A complex with the C-terminal portion of the proteosomal subunit S6 ATPase is the only available structure of a protein–protein interaction involving the oncoprotein gankyrin. However, difficulties associated with recombinant expression of S6 ATPase alone, or truncations thereof, have limited our understanding of this assembly. We replaced the C-terminal portion of FtsH from Escherichia coli with the structurally homologous C-terminal portion of S6 ATPase and used this grafted protein to characterize the gankyrin–S6 ATPase binding interaction by isothermal titration calorimetry.

Alanine scanning mutagenesis of a recently reported prostate cancer cell-selective Protein Transduction Domain (PTD) was used to assess the specific contribution each residue plays in cell uptake efficiency and cell-selectivity. These studies resulted in the identification of two key residues. Extensive mutagenesis at these key residues generated multiple mutants with significantly improved uptake efficiency and cell-selectivity profiles for targeted cells. The best mutant exhibits ∼19-fold better uptake efficiency and ∼4-fold improved cell-selectivity for a human prostate cancer cell line. In addition, while the native PTD sequence was capable of delivering functional fluorescent protein to the interior of a prostate cancer cells, only modest functional enzyme delivery was achieved. In contrast, the most potent mutant was able to deliver large quantities of a functional enzyme to the interior of human prostate cancer cells. Taken together, the research described herein has significantly improved the efficiency, cell-selectivity, and functional utility of a prostate cancer PTD.

DNA-mediated catalysis is an emerging field in bioorganic chemistry and chemical biology. However, the functional group diversity and known reactivity of DNA (A, T, C, and G) is relatively limited in scope. This relatively defined reactivity can limit the utility of DNA as a catalyst. In an effort to expand the functional group diversity and chemical reactivity of DNA, we sought to explore reactions involving single-stranded DNA equipped with a stabilized variant of N7-methyl guanine (2′-fluoro-5′-N7-methyl guanine). Here, we show that 5′-capped 2′-fluoro-N7-methyl guanine-labeled single-stranded DNA reacts with a ketone to afford a ketone-labeled DNA. This reaction likely proceeds through a reactive ylide or N-heterocyclic carbene. Taken together, our findings suggest that 2′-fluoro-5′-N7-methyl guanine is a stable adduct that can be selectively incorporated into ssDNA and functionalized with a ketone moiety by reaction with a simple ketone. Incorporation of this nucleoside into ssDNA may be useful for the evolution of novel deoxyribozymes that catalyze new reactions, including those which proceed via a reactive ylide or N-heterocyclic carbene-mediated chemistry.

Protein transduction domains (PTDs) are reagents that facilitate the delivery of diverse cargo to the interior of mammalian cells. We identified a PTD called “Ypep” (N-YTFGLKTSFNVQ-C), with cell penetration selectivity and potency profiles that are tightly controlled by multivalency effects. Pentavalent display of Ypep on M13 bacteriophage enables selective uptake of this phage in PC-3 human prostate cancer cells at low picomolar concentration and in the presence of human blood. All Ypep-dependent delivery is nontoxic and proceeds through energy-dependent endocytosis. Collectively, our results establish Ypep-displaying phage as a cell-penetrating platform with selectivity and potency profiles that compare to, or exceed, antibodies and their fragments. Our findings may have broader implications on the design of PTD technologies generated from phage display, as well as the use of Ypep-displaying phage as a prostate cancer cell-selective delivery platform.Highlights► Transduction domain potently and selectively penetrates PC-3 prostate cancer cells ► Bacteriophage selectively penetrate PC-3 prostate cancer cells at pM concentrations ► Potency and selectivity of PC-3 cell uptake is dependent upon multivalency effects

We report the in vitro selection of a single-stranded 72-nucleotide DNA enzyme (deoxyribozyme) that catalyzes a Friedel–Crafts reaction between an indole and acyl imidazole in good yield and in aqueous solvent. Appreciable Friedel–Crafts product requires addition of copper nitrate and the deoxyribozyme. We observe deoxyribozyme-mediated bond formation for both in cis and in trans Friedel–Crafts reactions.

U1A binds U1hpII, a hairpin RNA with a 10-nucleotide loop. A U1A mutant (ΔK50ΔM51) binds U1hpII-derived hairpins with shorter loops, making it an interesting scaffold for engineering or evolving proteins that bind similarly sized disease-related hairpin RNAs. However, a more detailed understanding of complexes involving ΔK50ΔM51 is likely a prerequisite to generating such proteins. Toward this end, we measured mutational effects for complexes involving U1A ΔK50ΔM51 and U1hpII-derived hairpin RNAs with seven- or eight-nucleotide loops and identified contacts that are critical to the stabilization of these complexes. Our data provide valuable insight into sequence-selective recognition of seven- or eight-nucleotide loop hairpins by an engineered RNA binding protein.

Gaining control over cell adhesion and growth is a critical step in microscale tissue engineering, as well as biosensor fabrication, applied cell biology, and high-throughput cell-based screening. Control over cell adhesion and growth is typically achieved by patterning small molecule or macromolecule reagents with affinity for a cell surface receptor onto a non-adhesive surface. These reagents are often susceptible to environmental and/or enzymatic degradation and their preparation and purification increase the overall cost and complexity of surface fabrication. Surface topology can influence cell adhesion and growth; however, engineering a surface with well-defined topology typically requires expensive and/or specialized equipment and/or multi-step processes such as microcontact printing. In this Paper we show that cell-imprinted features on the surface of a polyacrylamide hydrogel act as surface contact cues that promote cell adhesion and growth. In some cases the shape of cell-imprints dramatically affect cell adhesion. Collectively, we demonstrate that cell-imprinting polyacrylamide hydrogels is an inexpensive and straightforward method for programming cell adhesion and growth.

Split-GFP reassembly is an operationally simple in vivo technique used to identify and study interactions involving proteins and/or peptides. However, the instability of split-GFP fragments and their susceptibility to aggregation place limitations on the broader use of split-GFP reassembly. Supercharged proteins, including supercharged GFP, are variants with high theoretical negative or positive charge that are resistant to aggregation. We show that a split-superpositive GFP (split-spGFP) variant reassembles faster and more efficiently than previously reported split-sg100 GFP and split-folding-reporter GFP (split-frGFP) systems. In addition, interaction-dependent split-spGFP reassembly is efficient at physiological temperature. The increased efficiency and robustness of split-spGFP reassembly make this reporter system ideal for identifying and studying interactions involving proteins and/or peptides in vivo, and may be particularly useful for identifying or studying interactions involving proteins or peptides that are themselves susceptible to aggregation.

We report a one-pot synthesis of well-defined micron-sized or nanoscale poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) particles. We show that these pre-activated particles can be easily coupled to an amine-functionalized cargo in a single step to generate particles with uniform fluorescence.

A maximally divergent “build–couple” synthesis of heterofunctionalized polyvalent molecules is described. This strategic approach enables the synthesis of highly diverse polyvalent structures from a pre-programmed combinatorial set of modules.

A maximally divergent “build–couple” synthesis of heterofunctionalized polyvalent molecules is described. This strategic approach enables the synthesis of highly diverse polyvalent structures from a pre-programmed combinatorial set of modules.

We report the in vitro selection of a single-stranded 72-nucleotide DNA enzyme (deoxyribozyme) that catalyzes a Friedel–Crafts reaction between an indole and acyl imidazole in good yield and in aqueous solvent. Appreciable Friedel–Crafts product requires addition of copper nitrate and the deoxyribozyme. We observe deoxyribozyme-mediated bond formation for both in cis and in trans Friedel–Crafts reactions.