Matthew Levy

Synthetic biology: engineering Escherichia coli to see light.

We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to ‘print’ complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.

Aptamer mediated siRNA delivery

Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.

Quantum‐Dot Aptamer Beacons for the Detection of Proteins

Quantum dots (QDs) offer a number of advantages over standard fluorescent dyes for monitoring biological systems in real time, including greater photostability, larger effective Stokes shifts, longer fluorescent lifetimes, and sharper emission bands than traditional organic fluorophores. In addition, QDs all respond to the same excitation wavelength, but emit at different wavelengths; this should allow for the multiplex detection of different analytes in parallel. Recent work has also demonstrated that QDs can be used in the construction of biosensors that signal by fluorescence resonance energy transfer (FRET). For example, a quantum-dotbased molecular beacon has been described in which the nonfluorescent dye DABCYL was used to quench the fluorescence of the quantum dot. In the presence of a target DNA, the opening of the molecular beacon resulted in an approximately fivefold increase in fluorescence of the quantum dot. Similarly, a QD-based protein biosensor that can detect the small molecule maltose has been designed. In one configuration of this system, a maltose-binding protein was complexed to the ZnS shell of a CdSe QD through a 5-histidine tail appended to the protein. Binding of a dye-labeled cyclodextrin molecule to the protein resulted in a loss of photoluminescence, which could be restored by the displacement of the bound cyclodextrin by the addition of maltose. We have now designed a quantum-dot-based aptamer biosensor. Like molecular beacons, aptamer beacons rely on analyte-dependent conformational changes that can alter the proximity of optical reporters to one another, and thus can potentially alter the fluorescence resonance energy transfer (FRET) between these reporters. Aptamer beacons have previously been designed for a variety of targets including small molecules, such as ATP and cocaine, and protein targets, such as PDGF, thrombin, and the HIV-1 Tat. We utilized an aptamer that is known to bind the blood-clotting protein thrombin as a model system. While thrombin aptamer beacons that utilize organic fluorophores have previously been developed, the adaptation of these strategies to the development of a quantum-dot aptamer beacon (QDB) was not straightforward, primarily because the inorganic quantum dot is much larger and brighter than its organic partner. The development of quantum-dot beacons therefore relied upon two important design features. First, in order to ensure that a substantive conformational change would occur upon analyte binding, we synthesized a quantum-dot beacon based on a two-piece aptamer beacon originally developed by Li et al. in 2002. The final construct consisted of the anti-thrombin aptamer conjugated to a quantum dot (Figure 1A; underlined), and an oligonucleotide

Biocompatible copper(I) catalysts for in vivo imaging of glycans.

The Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is the standard method for bioorthogonal conjugation. However, current Cu(I) catalyst formulations are toxic, hindering their use in living systems. Here we report that BTTES, a tris(triazolylmethyl)amine-based ligand for Cu(I), promotes the cycloaddition reaction rapidly in living systems without apparent toxicity. This catalyst allows, for the first time, noninvasive imaging of fucosylated glycans during zebrafish early embryogenesis. We microinjected embryos with alkyne-bearing GDP-fucose at the one-cell stage and detected the metabolically incorporated unnatural sugars using the biocompatible click chemistry. Labeled glycans could be imaged in the enveloping layer of zebrafish embryos between blastula and early larval stages. This new method paves the way for rapid, noninvasive imaging of biomolecules in living organisms.

Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation

Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When ≈800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution.

Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer−gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(−) cell lines treated with the anti-PSMA aptamer−gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications.

Exponential growth by cross-catalytic cleavage of deoxyribozymogens

We have designed an autocatalytic cycle based on the highly efficient 10–23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10–23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an in vitro selection experiment conducted in the absence of any protein enzymes.

Aptamers as potential tools for super-resolution microscopy

(a) The TfnR aptamer c2, the EGFR aptamer E07 and their respective control aptamers (random sequences) were incubated at 37°C with human A431 cells as described in Supplementary Methods. Similarly, HeLa cells stably transfected with a human PSMA construct were incubated with the PSMA A9 aptamer or its random control. The pairs of images (control and aptamer) are equally scaled to allow a direct visual comparison. The insets in the control images correspond to the same images, scaled to a level where autofluorescence can be visualized. Scale bar, 10 μm. (b) Colocalization of the different aptamers with endosomal labels. We co-incubated the cells (same as above) with aptamers against TfnR (c2) or PSMA (A9) and Alexa488-transferrin (Invitrogen), since the latter constitutes an ideal marker for early endosomes.

Aptamers and aptamer targeted delivery

When aptamers first emerged almost two decades ago, most were RNA species that bound and tagged or inhibited simple target ligands. Very soon after, the ‘selectionologists’ developing aptamer technology quickly realized more potential for the aptamer. In recent years, advances in aptamer techniques have enabled the use of aptamers as small molecule inhibitors, diagnostic tools and even therapeutics. Aptamers are now being employed in novel applications. We review, herein, some of the recent and exciting applications of aptamers in cell-specific recognition and delivery.

An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.