David H. J. Bunka

Aptamers come of age – at last

Nucleic-acid aptamers have the molecular recognition properties of antibodies, and can be isolated robotically for high-throughput applications in diagnostics, research and therapeutics. Unlike antibodies, however, they can be chemically derivatized easily to extend their lifetimes in biological fluids and their bioavailability in animals. The first aptamer-based clinical drugs have recently entered service. Meanwhile, active research programmes have identified a wide range of anti-viral aptamers that could form the basis for future therapeutics.

Toggled RNA Aptamers Against Aminoglycosides Allowing Facile Detection of Antibiotics Using Gold Nanoparticle Assays

We have used systematic evolution of ligands by exponential enrichment (SELEX) to isolate RNA aptamers against aminoglycoside antibiotics. The SELEX rounds were toggled against four pairs of aminoglycosides with the goal of isolating reagents that recognize conserved structural features. The resulting aptamers bind both of their selection targets with nanomolar affinities. They also bind the less structurally related targets, although they show clear specificity for this class of antibiotics. We show that this lack of aminoglycoside specificity is a common property of aptamers previously selected against single compounds and described as “specific”. Broad target specificity aptamers would be ideal for sensors detecting the entire class of aminoglycosides. We have used ligand-induced aggregation of gold-nanoparticles coated with our aptamers as a rapid and sensitive assay for these compounds. In contrast to DNA aptamers, unmodified RNA aptamers cannot be used as the recognition ligand in this assay, whereas 2′-fluoro-pyrimidine derivatives work reliably. We discuss the possible application of these reagents as sensors for drug residues and the challenges for understanding the structural basis of aminoglycoside-aptamer recognition highlighted by the SELEX results.

Production and Characterization of RNA Aptamers Specific for Amyloid Fibril Epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-β architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloid-like fibrils formed in vitro from β2-microglobulin (β2m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent KD ∼ nm) to β2m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of β2m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.

Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein.

BackgroundDefects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.ResultsWe determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.ConclusionIt is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.

An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein

Background Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.

Effects of single nucleotide changes on the binding and activity of RNA aptamers to human papillomavirus 16 E7 oncoprotein.

A virally-encoded oncoprotein (E7 from human papillomavirus 16, involved in the initiation of cell transformation) was the target for RNA aptamer development by the process of systematic evolution of ligands by exponential enrichment (SELEX). A number of aptamers were identified, one of which was shown to inhibit the interaction between E7 and its major binding partner, pRb. Aptamers with very similar sequences (more than 92% similarity in the random regions) did not share this activity. This study demonstrates the potential of aptamers to be highly specific, with small differences in aptamer sequence having profound effects on function.

Generation of neutralizing aptamers against herpes simplex virus type 2: potential components of multivalent microbicides.

The prophylactic use of topical antiviral agents has recently been validated by the reduction in human immunodeficiency virus (HIV) type 1 infection incidence seen using tonofovir-containing microbicides. In order to develop a wide-spectrum microbicide to prevent infection with a wide range of sexually transmitted viruses, we have previously reported the development of HIV-neutralizing aptamers and here report the isolation and characterization of aptamers that neutralize herpes simplex virus type 2 (HSV-2). These aptamers bind the envelope glycoprotein (gD), are potent (IC(50) of 20-50 nM) and are able to block infection pathways dependent on both major entry receptors, Nectin1 and HVEM. Structural analysis and mutagenesis of these aptamers reveal a core specificity element that could provide the basis for pharmaceutical development. As HSV-2 is a major risk factor for the acquisition of HIV-1, a microbicide capable of preventing HSV-2 infection would not only reduce the morbidity associated with HSV-2, but also that derived from HIV-1.

Distinguishing closely-related amyloid precursors using an RNA aptamer

Background: Altering the co-polymerization of proteins into amyloid fibrils provides an opportunity for manipulating fibril assembly. Results: NMR and kinetic analysis showed that an RNA aptamer distinguishes between two highly similar co-aggregating proteins. Conclusion: RNA aptamers are specific and discriminatory probes able to modulate amyloid formation. Significance: Aptamers can be used as tools to differentiate amyloid precursors that are closely related and alter assembly. Although amyloid fibrils assembled in vitro commonly involve a single protein, fibrils formed in vivo can contain multiple protein sequences. The amyloidogenic protein human β2-microglobulin (hβ2m) can co-polymerize with its N-terminally truncated variant (ΔN6) in vitro to form hetero-polymeric fibrils that differ from their homo-polymeric counterparts. Discrimination between the different assembly precursors, for example by binding of a biomolecule to one species in a mixture of conformers, offers an opportunity to alter the course of co-assembly and the properties of the fibrils formed. Here, using hβ2m and its amyloidogenic counterpart, ΔΝ6, we describe selection of a 2′F-modified RNA aptamer able to distinguish between these very similar proteins. SELEX with a N30 RNA pool yielded an aptamer (B6) that binds hβ2m with an EC50 of ∼200 nm. NMR spectroscopy was used to assign the 1H-15N HSQC spectrum of the B6-hβ2m complex, revealing that the aptamer binds to the face of hβ2m containing the A, B, E, and D β-strands. In contrast, binding of B6 to ΔN6 is weak and less specific. Kinetic analysis of the effect of B6 on co-polymerization of hβ2m and ΔN6 revealed that the aptamer alters the kinetics of co-polymerization of the two proteins. The results reveal the potential of RNA aptamers as tools for elucidating the mechanisms of co-assembly in amyloid formation and as reagents able to discriminate between very similar protein conformers with different amyloid propensity.

CHAPTER 6:Therapeutic Applications of Nucleic Acid Aptamer Conjugates

Nucleic acid aptamers can be isolated by in vitro techniques (SELEX), and they recognise a wide variety of molecular targets with affinities and specificities that rival those of antibodies. Unlike those protein-based ligands, aptamers are easily produced by enzymatic or chemical means, are readily modified for incorporation of additional functionalities or formats, and have more predictable physical properties. One application of this technology is the development of therapeutic aptamers. This chapter will describe some of the latest advances in this field, highlighting particularly the innovative ways in which aptamers are being adapted to therapy.