Amy Yan

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.

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.

Crystal Structure and RNA Binding Properties of the RNA Recognition Motif (RRM) and AlkB Domains in Human AlkB Homolog 8 (ABH8), an Enzyme Catalyzing tRNA Hypermodification

Background: Human ABH8 is a tRNA-hypermodifying enzyme paralogous to the DNA repair enzyme AlkB. Results: Crystal structures of the RRM/AlkB domains of ABH8 were determined in conjunction with thermodynamic assays. Conclusion: Substrate specificity and catalytic activity are modulated by conformational adaptations in and around the active site. Significance: These results provide insight into the functional expansion of the AlkB enzyme family in higher eukaryotes. Humans express nine paralogs of the bacterial DNA repair enzyme AlkB, an iron/2-oxoglutarate-dependent dioxygenase that reverses alkylation damage to nucleobases. The biochemical and physiological roles of these paralogs remain largely uncharacterized, hampering insight into the evolutionary expansion of the AlkB family. However, AlkB homolog 8 (ABH8), which contains RNA recognition motif (RRM) and methyltransferase domains flanking its AlkB domain, recently was demonstrated to hypermodify the anticodon loops in some tRNAs. To deepen understanding of this activity, we performed physiological and biophysical studies of ABH8. Using GFP fusions, we demonstrate that expression of the Caenorhabditis elegans ABH8 ortholog is widespread in larvae but restricted to a small number of neurons in adults, suggesting that its function becomes more specialized during development. In vitro RNA binding studies on several human ABH8 constructs indicate that binding affinity is enhanced by a basic α-helix at the N terminus of the RRM domain. The 3.0-Å-resolution crystal structure of a construct comprising the RRM and AlkB domains shows disordered loops flanking the active site in the AlkB domain and a unique structural Zn(II)-binding site at its C terminus. Although the catalytic iron center is exposed to solvent, the 2-oxoglutarate co-substrate likely adopts an inactive conformation in the absence of tRNA substrate, which probably inhibits uncoupled free radical generation. A conformational change in the active site coupled to a disorder-to-order transition in the flanking protein segments likely controls ABH8 catalytic activity and tRNA binding specificity. These results provide insight into the functional and structural adaptations underlying evolutionary diversification of AlkB domains.

A General RNA Motif for Cellular Transfection

We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.

Cell internalization SELEX: In vitro selection for molecules that internalize into cells

Aptamer technology allows for the selection of nucleic acids that can bind to and enter cells. By establishing conditions during the selection that eliminate cell-surface binders as well as non-internalizing RNAs, only extremely tightly bound aptamers or aptamers that have internalized are recovered. We describe a general scheme for selecting RNA molecules that are capable of internalizing into cells and discuss the factors that can affect a successful selection. Much like standard cell-surface selections, these types of selections should be possible independent of detailed knowledge of the cell surface.

Erratum: An RNA alternative to human transferrin: A new tool for targeting human cells (Molecular Therapy - Nucleic Acids (2013) 2 e79 Doi:10.1038/mtna. 2013.6)

1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York, USA; 2Universidade do Estado de Santa Catarina, Centro de Ciências Agroveterinárias, Lages, Santa Catarina, Brazil; 3Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas, USA; 4STED Microscopy of Synaptic Function, European Neuroscience Institute, Göttingen, Germany. Matthew Levy 1301 Morris Park Avenue, Price Center, Room 519, Bronx, NY, 10461, USA. Correspondence: E-mail: matthew.levy@einstein.yu.edu Received 11 November 2011; revised 2 March 2012; accepted 26 March 2012

A Hitchhiker's Guide to HIV

Although anti-HIV drugs have been in place for decades, both prophylaxis and a cure remain elusive. Many treatments for HIV infection are associated with severe side effects (which may contribute to poor patient compliance), and resistance mutants frequently arise that render many front-line therapeutics impotent.

144. In Vivo Targeting hTfR and EGFR Using Aptamers

Aptamers which bind cell surface receptors represent an exciting and potentially important class of ligands for the development of diagnostics and therapeutics. However, if aptamers are to be utilized in vivo, and more importantly for the targeted delivery of therapeutic cargoes, the molecular function must be robust, especially with regard to specificity and activity. Because assay formats for determining the binding specificity of aptamers to their cell surface targets vary greatly, and because some molecules have yet to be tested in vivo, it remains difficult to compare the function of these molecules and to identify which candidate molecules could be used for future development as both diagnostics and therapeutics. In order to assess this, we have chemically synthesized 15 different aptamers from the literature which target a range of human cell surface receptors associated with cancers. These include aptamers targeting EGFR, EPCAM, nucleolin, PSMA, PTK7 and hTfR, as well as some aptamers which have been derived from variations on ‘whole cell SELEX’ or similar approaches against cancer cells lines. Using standardized conditions, we assayed each aptamer in vitro using flow cytometry on a panel of different cancer cell lines to compare relative binding affinity and specificity. Molecules which function in vitro were subsequently assessed for tumor targeting capabilities in vivo using near-infrared imaging. Interestingly, some molecules which function robustly in vitro demonstrate little to no activity in vivo. Of the molecules tested,three demonstrate significant activity in vivo. These include an anti-EGFR aptamer, an anti-hTfR aptamer and a molecule identified via an ‘internalization selection method’ that is readily internalized by cancer cells, but shows little uptake by normal tissue. Taken as a whole, our data demonstrate some surprising differences in the apparent affinities and specificities of different aptamers when assayed under these standardized conditions. They also provide us with molecules which we can now think about transitioning for in vivo applications. Data on using these molecules to deliver cytotoxic drugs in vitro and in vivo will be presented.