Zbigniew Pianowski

Imaging of mRNA in Live Cells Using Nucleic Acid-Templated Reduction of Azidorhodamine Probes

Nucleic acid-templated reactions leading to a fluorescent product represent an attractive strategy for the detection and imaging of cellular nucleic acids. Herein we report the use of a Staudinger reaction to promote the reduction of profluorescent azidorhodamine. The use of two cell-permeable GPNA probes, one labeled with the profluorescent azidorhodamine and the other with trialkylphosphine, enabled the detection of the mRNA encoding O-6-methylguanine-DNA methyltransferase in intact cells.

Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis

Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.

Efficient in Vitro Encapsulation of Protein Cargo by an Engineered Protein Container

An engineered variant of lumazine synthase, a nonviral capsid protein with a negatively charged luminal surface, is shown to encapsulate up to 100 positively supercharged green fluorescent protein (GFP) molecules in vitro. Packaging can be achieved starting either from intact, empty capsids or from capsid fragments by incubation with cargo in aqueous buffer. The yield of encapsulated GFP correlates directly with the host/guest mixing ratio, providing excellent control over packing density. Facile in vitro loading highlights the unusual structural dynamics of this novel nanocontainer and should facilitate diverse biotechnological and materials science applications.

Fluorescence-based detection of single nucleotide permutation in DNA via catalytically templated reaction.

Templated reduction of low fluorescence azidocoumarin-PNA conjugate to high fluorescence aminocoumarin was achieved using a catalytic amount of DNA with single nucleotide resolution.

Exploration of Alternate Catalytic Mechanisms and Optimization Strategies for Retroaldolase Design

Designed retroaldolases have utilized a nucleophilic lysine to promote carbon-carbon bond cleavage of β-hydroxy-ketones via a covalent Schiff base intermediate. Previous computational designs have incorporated a water molecule to facilitate formation and breakdown of the carbinolamine intermediate to give the Schiff base and to function as a general acid/base. Here we investigate an alternative active-site design in which the catalytic water molecule was replaced by the side chain of a glutamic acid. Five out of seven designs expressed solubly and exhibited catalytic efficiencies similar to previously designed retroaldolases for the conversion of 4-hydroxy-4-(6-methoxy-2-naphthyl)-2-butanone to 6-methoxy-2-naphthaldehyde and acetone. After one round of site-directed saturation mutagenesis, improved variants of the two best designs, RA114 and RA117, exhibited among the highest kcat (>10(-3)s(-1)) and kcat/KM (11-25M(-1)s(-1)) values observed for retroaldolase designs prior to comprehensive directed evolution. In both cases, the >10(5)-fold rate accelerations that were achieved are within 1-3 orders of magnitude of the rate enhancements reported for the best catalysts for related reactions, including catalytic antibodies (kcat/kuncat=10(6) to 10(8)) and an extensively evolved computational design (kcat/kuncat>10(7)). The catalytic sites, revealed by X-ray structures of optimized versions of the two active designs, are in close agreement with the design models except for the catalytic lysine in RA114. We further improved the variants by computational remodeling of the loops and yeast display selection for reactivity of the catalytic lysine with a diketone probe, obtaining an additional order of magnitude enhancement in activity with both approaches.

Probing Biology with Small Molecule Microarrays (SMM)

In the continuous drive to increase screening throughput and reduce sample requirement, microarray-based technologies have risen to the occasion. In the past 7 years, a number of new methodologies have been developed for preparing small molecule microarrays from combinatorial and natural product libraries with the goal of identifying new interactions or enzymatic activities. Recent advances and applications of small molecule microarrays are reviewed.

Selection of bead-displayed, PNA-encoded chemicals

The lack of efficient identification and isolation methods for specific molecular binders has fundamentally limited drug discovery. Here, we have developed a method to select peptide nucleic acid (PNA) encoded molecules with specific functional properties from combinatorially generated libraries. This method consists of three essential stages: (1) creation of a Lab‐on‐Bead™ library, a one‐bead, one‐sequence library that, in turn, displays a library of candidate molecules, (2) fluorescence microscopy‐aided identification of single target‐bound beads and the extraction – wet or dry – of these beads and their attached candidate molecules by a micropipette manipulator, and (3) identification of the target‐binding candidate molecules via amplification and sequencing. This novel integration of techniques harnesses the sensitivity of DNA detection methods and the multiplexed and miniaturized nature of molecule screening to efficiently select and identify target‐binding molecules from large nucleic acid encoded chemical libraries. Beyond its potential to accelerate assays currently used for the discovery of new drug candidates, its simple bead‐based design allows for easy screening over a variety of prepared surfaces that can extend this techniques application to the discovery of diagnostic reagents and disease markers. Copyright

Substrate screening identifies a novel target sequence for the proteasomal activity regulated by ionizing radiation

The screening for treatment‐induced enzyme activities offers the opportunity to discover important regulatory mechanisms and the identification of potential targets for anti‐cancer therapies. A novel screening technique was applied to screen substrate peptide sequences for proteolytic activities up‐ or down‐regulated by ionizing radiation in tumor cells. One specific substrate sequence was cleaved in control cell extracts but to a smaller extent in irradiated cell extracts and investigated in detail. Based on protease‐class‐specific inhibitory studies and cleavage site analysis a potent warhead‐inhibitor was synthesized and used to identify the proteasome as the protease of interest. The investigated sequence shows high homology to a regulatory site of nucleoporin 50, an element of the nuclear pore complex, and site specific cleavage of nucleoporin 50 was determined in vitro suggesting a novel link between the ionizing radiation‐regulated proteasome and nuclear protein shuttling.

Photocontrol of Drug Release from Supramolecular Hydrogels with Green Light

Photoresponsive smart materials transform light energy into sophisticated functions. They find increasing biomedical applications in light-induced drug-release and photopharmacology, because they can provide the desired therapeutic effect locally due to precise spatiotemporal dosage control. However, the majority of reported studies rely on cytotoxic UV light that penetrates tissues poorly. Here, we report the first drug-releasing system based on photochromic low molecular weight supramolecular hydrogels that is triggered with visible light. We demonstrated green-light-induced release of structurally unmodified antibiotic, anticancer, and anti-inflammatory drugs under physiological conditions. Using the antibiotic-loaded gel, we selectively inhibited bacterial growth with green light.