RNA aptamers specific for bovine thrombin

Bovine thrombin is widely used in clinical wound healing after surgery. There is 85% homology between bovine thrombin and human thrombin, so most antibodies against bovine thrombin cross-react with human thrombin. Rare antibodies against bovine thrombin but not cross-reacting with human thrombin have been reported. RNA ligands (aptamers) have been used to bind to target molecules with sometimes higher specificity than antibodies. Here we report the isolation of aptamers specific for bovine thrombin by systematic evolution of ligands by exponential enrichment (SELEX) from an RNA pool containing a 25-nucleotide randomized region. After seven rounds of selection, two aptamers specific for bovine thrombin were identified with a K(d) of 164 and 240 nM, respectively. Significantly, these aptamers do not bind to human thrombin. Secondary structure prediction revealed potential stem-loop structures for these RNAs. Both RNA aptamers inhibit only bovine thrombin-catalyzed fibrin clot formation in vitro. Competition assay results suggested that the RNA aptamers might bind to the electropositive domain of bovine thrombin, that is, heparin-binding site, instead of fibrinogen-recognition exosite. The resulting bovine-specific thrombin inhibitor might be used in some clinical applications when bovine thrombin activity needs to be contained or in research where human and bovine thrombin need to be distinguished.     

Production and characterization of RNA aptamers specific for amyloid fibril epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-beta 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 beta(2)-microglobulin (beta(2)m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K(D) approximately nm) to beta(2)m 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 beta(2)m. 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.     

Selection of RNA aptamers specific to active prostate-specific antigen

A counter-SELEX procedure with recombinant purified active prostate specific antigen (PSA) was used to identify specific RNA aptamers against the active PSA. We developed two different kinds of counter-SELEX methods; one includes pre-clearance step with inactive proPSA protein, and the other with tagged GST protein. After 9 iterative selection cycles, several identical RNA aptamers can be identified from both counter-SELEX methods. Real-time PCR analysis and gel retardation experiment showed that the aptamers have a specific binding activity against the active PSA, but not for GST or proPSA. These aptamers could be of potential use as specific diagnostic, imaging and/or therapeutic agents against prostate cancer.     

Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells

The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2′-F substituted RNA aptamers that bind to the HIV-1_BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer–siRNA chimeras. The aptamers and aptamer–siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a ‘sticky’ sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.     

Molecular aptamers for drug delivery

The active targeting of drugs in a cell-, tissue- or disease-specific manner represents a potentially powerful technology with widespread applications in medicine, including the treatment of cancers. Aptamers have properties such as high affinity and specificity for targets, easy chemical synthesis and modification, and rapid tissue penetration. They have become attractive molecules in diagnostics and therapeutics rivaling and, in some cases, surpassing other molecular probes, such as antibodies. In this review, we highlight the recent progress in aptamer-mediated delivery for therapeutics and disease-targeting based on aptamer integration with a variety of nanomaterials, such as gold nanorods, DNA micelles, DNA hydrogels and carbon nanotubes.     

Improved methods for the delivery of liposome-sequestered RNA into eucaryotic cells

RNA sequestered by negatively charged liposomes becomes cell-associated following interaction between eucaryotic cells and the liposomes. This paper provides evidence that cell-associated RNA is internalized by the cells. In fact, (a) when Escherichia coli and mammalian RNA are entrapped within the same liposome population and delivered into cultured cells, one can observe degradation of the procaryotic but not the eucaryotic RNA. Such an event cannot happen extracellularly. (b) Scanning electron microscopy reveals no more than 10 liposomes adhering to each cell upon liposome-cell interaction under conditions in which the RNA entrapped by 140 liposomes becomes associated with each cell. The ability of liposomes prepared by (a) the cochleate process, (b) the reverse-phase evaporation technique, and (c) the ether infusion technique, to sequester and deliver RNA into cells was investigated. Reverse-phase evaporated liposomes were most efficient in sequestering RNA (20–40%), however, all types of liposomes delivered RNA with comparable efficiency. The rate of liposome-mediated RNA delivery into mammalian cells could be substantially improved when: (a) liposome-cell interaction was carried out at pH 6.5 (twofold increase over pH 7.5), (b) a basic protein (methylated albumin) was present (two- to threefold increase), (c) liposome-cell cultures were treated with polyethylene glycol 6000 (four- to eight-fold increase), and (d) DEAE-dextran was added during interaction of liposomes with cell monolayers (four- to eight-fold increase).     

In vitro selection of specific RNA aptamers for the NFAT DNA binding domain

Nuclear factor of activated T cells (NFAT) plays a central role in the immune response, and the immuno-suppressive drugs, cyclosporin A and FK-506, have been developed to inhibit it. However, due to the toxic effects of these drugs, which derive from their ability to inhibit calcineurin in non-immune tissues, the identification of small compounds that target NFAT directly could be an approach to developing less toxic immunosuppressive therapy. Using an in vitro selection technology termed SELEX on a combinatorial RNA library with 40 nucleotide-long random sequences, we have isolated two RNA aptamers to the NFAT DNA binding domain (DBD). Gel retardation assays and surface plasmon resonance measurements showed that the aptamers have a specific and high affinity (apparent KD~10 to 100 nM) for the NFAT DBD. Enzymatic probing analysis showed that the two RNA aptamers have similar structures and share a sequence that forms an apical loop. Moreover, RNase footprinting analysis showed that the shared sequence (GATATGAAGGA/ TGTG/AGAGAG) is critical for binding to both NFATp DBD and NFATc DBD. These results suggest that short RNAs identified in this study is a specific aptamer to NFAT DBD, and hence could be applied not only for the delineation of NFAT functions but for the development of potent immune modulating lead compounds.     

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.     

Enhancing Interleukin-6 and Interleukin-11 receptor cleavage

Proteolytic cleavage of the membrane-bound Interleukin-6 receptor (IL-6R) by the metalloprotease ADAM17 releases an agonistic soluble form of the IL-6R (sIL-6R), which is responsible for the pro-inflammatory trans-signaling branch of the cytokine's activities. This proteolytic step, which is also called ectodomain shedding, is critically regulated by the cleavage site within the IL-6R stalk, because mutations or small deletions within this region are known to render the IL-6R irresponsive towards proteolysis. In the present study, we employed cleavage site profiling data of ADAM17 to generate an IL-6R with increased cleavage susceptibility. Using site-directed mutagenesis, we showed that the non-prime sites P3 and P2 and the prime site P1′ were critical for this increase in proteolysis, whereas other positions within the cleavage site were of minor importance. Insertion of this optimized cleavage site into the stalk of the Interleukin-11 receptor (IL-11R) was not sufficient to enable ADAM17-mediated proteolysis, but transfer of different parts of the IL-6R stalk enabled shedding by ADAM17. These findings shed light on the cleavage site specificities of ADAM17 using a native substrate and reveal further differences in the proteolysis of IL-6R and IL-11R.     

RNA aptamers selected against the GluR2 glutamate receptor channel

The excessive activation of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, a subtype of glutamate ion channels, has been implicated in various neurological diseases such as cerebral ischemeia and amyotrophic lateral sclerosis. Inhibitors of AMPA receptors are drug candidates for potential treatment of these diseases. Using the systematic evolution of ligands by exponential enrichment (SELEX), we have selected a group of RNA aptamers against the recombinant GluR2Qflip AMPA receptor transiently expressed in HEK-293 (human embryonic kidney) cells. One of the aptamers, AN58, is shown to competitively inhibit the receptor. The nanomolar affinity of AN58 rivals that of NBQX (6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione), one of the best competitive inhibitors. Like NBQX, AN58 has the highest affinity for GluR2, the selection target, among all AMPA receptor subunits. However, AN58 has a higher selectivity for the GluR4 AMPA receptor subunit and remains potent even at pH = 6.8 (i.e., a clinically relevant acidic pH), as compared with NBQX. Furthermore, this RNA molecule possesses stable physical properties. Therefore, AN58 serves as a unique lead compound for developing water-soluble inhibitors with a nanomolar affinity for GluR2 AMPA receptors.     

Interleukin-6: a new therapeutic target

The therapeutic success of biological agents, especially the tumour necrosis factor (TNF) inhibitors, has opened a new chapter in the book of therapies for rheumatoid arthritis. Nevertheless, more than 50% of patients may not respond by > 50% improvement. New compounds have recently entered the treatment arena. One of these is rituximab, which depletes B cells, and another, abatacept, interferes with T-cell co-stimulation. However, although these agents may be effective in a number of patients who fail to respond to TNF blockade, they only rarely induce remission and overall 50% response rates do not exceed those with the TNF inhibitors. Among the major proinflammatory cytokines, IL-6 plays a pleiotropic role both in terms of activating the inflammatory response and osteoclastogenesis. Here, we review recent phase II trials of tocilizumab, a humanized anti-IL-6 receptor antibody that achieves a significant therapeutic response rate.     

Serum-stable RNA aptamers to urokinase-type plasminogen activator blocking receptor binding

The serine proteinase urokinase-type plasminogen activator (uPA) is widely recognized as a potential target for anticancer therapy. Its association with cell surfaces through the uPA receptor (uPAR) is central to its function and plays an important role in cancer invasion and metastasis. In the current study, we used systematic evolution of ligands by exponential enrichment (SELEX) to select serum-stable 2'-fluoro-pyrimidine-modified RNA aptamers specifically targeting human uPA and blocking the interaction to its receptor at low nanomolar concentrations. In agreement with the inhibitory function of the aptamers, binding was found to be dependent on the presence of the growth factor domain of uPA, which mediates uPAR binding. One of the most potent uPA aptamers, upanap-12, was analyzed in more detail and could be reduced significantly in size without severe loss of its inhibitory activity. Finally, we show that the uPA-scavenging effect of the aptamers can reduce uPAR-dependent endocytosis of the uPA-PAI-1 complex and cell-surface associated plasminogen activation in cell culture experiments. uPA-scavenging 2'-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.     

A method for phospholipid-mediated delivery of specific antibodies into adherent cultured cells.

A simple and rapid procedure for the intracellular delivery of macromolecules into adherent cultured cells is described. Cells are incubated with cold glycerol, then transiently made permeable with L-alpha-lysophosphatidylcholine (LPC) in the presence of test compound to be loaded into cells. LPC induces temporary permeability of the plasma membrane, as evidenced by the loss and recovery of the cells' ability to exclude trypan blue. Molecules at least as large as antibodies are internalized during this transient permeability. Antibodies delivered intracellularly in this manner are able to complex with their specific antigen and exert functional consequences on normal cell metabolism, suggesting that this procedure is useful for determining protein function. As one example of this, we present data on the ability of specific antibodies, delivered intracellularly in this manner, to inhibit morphological differentiation (i.e., neurite outgrowth) in a neuroblastoma cell line.     

Generation of RNA aptamers to the G-protein-coupled receptor for neurotensin,NTS-1

G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.     

Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.     

Selection of RNA aptamers against mouse embryonic stem cells

Embryonic stem cells (ESCs) are capable of unlimited self-renewal and differentiation into multiple cell types. Recent large-scale analyses have identified various cell surface molecules on ESCs. Some of them are considered to be beneficial markers for characterization of cellular phenotypes and/or play an essential role for regulating the differentiation state. Thus, it is desired to efficiently produce affinity reagents specific to these molecules. In this study, to develop such reagents for mouse ESCs (mESCs), we selected RNA aptamers against intact, live mESCs using several selection strategies. The initial selection provided us with several anti-mESC aptamers of distinct sequences, which unexpectedly react with the same molecule on mESCs. Then, to isolate aptamers against different surface markers on mESCs, one of the selected aptamers was used as a competitor in the subsequent selections. In addition, one of the selections further employed negative selection against differentiated mouse cells. Consequently, we successfully isolated three classes of anti-mESC aptamers that do not compete with one another. The isolated aptamers were shown to distinguish mESCs from differentiated mouse cell lines and trace the differentiation process of mESCs. These aptamers could prove useful for developing molecular probes and manipulation tools for mESCs.     

Intracellular cargo delivery by an octaarginine transporter adapted to target prostate cancer cells through cell surface protease activation

Delivery of therapeutics and imaging agents to target tissues requires localization and activation strategies with molecular specificity. Cell-associated proteases can be used for these purposes in a number of pathologic conditions, and their enzymatic activities can be exploited for activation strategies. Here, molecules based on the d-arginine octamer (r8) protein-transduction domain (PTD, also referred to as molecular transporters) have been adapted for selective uptake into cells only after proteolytic cleavage of a PTD-attenuating sequence by the prostate-specific antigen (PSA), an extracellular protease associated with the surface and microenvironment of certain prostate cancer cells. Convergent syntheses of these activatable PTDs (APTDs) are described, and the most effective r8 PTD-attenuating sequence is identified. The conjugates are shown to be stable in serum, cleaved by PSA, and taken up into Jurkat (human T cells) and PC3M prostate cancer cell lines only after cleavage by PSA. These APTD peptide-based molecules may facilitate targeted delivery of therapeutics or imaging agents to PSA-expressing prostate cancers.