Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues

SDA, a DNA aptamer inhibiting E- and P-selectin mediated adhesion of cancer and leukemia cells, the first and pivotal step in transendothelial migration during metastasis formation.

Endothelial (E-) and platelet (P-) selectin mediated adhesion of tumor cells to vascular endothelium is a pivotal step of hematogenous metastasis formation. Recent studies have demonstrated that selectin deficiency significantly reduces metastasis formation in vivo. We selected an E- and P-Selectin specific DNA Aptamer (SDA) via SELEX (Systematic Evolution of Ligands by EXponential enrichment) with a K(d) value of approximately 100 nM and the capability of inhibiting the interaction between selectin and its ligands. Employing human colorectal cancer (HT29) and leukemia (EOL-1) cell lines we could demonstrate an anti-adhesive effect for SDA in vitro. Under physiological shear stress conditions in a laminar flow adhesion assay, SDA inhibited dynamic tumor cell adhesion to immobilized E- or P-selectin. The stability of SDA for more than two hours allowed its application in cell-cell adhesion assays in cell culture medium. When adhesion of HT29 cells to TNFα-stimulated E-selectin presenting human pulmonary microvascular endothelial cells was analyzed, inhibition via SDA could be demonstrated as well. In conclusion, SDA is a potential new therapeutic agent that antagonizes selectin-mediated adhesion during metastasis formation in human malignancies

Adhesion of circulating tumor cells towards vascular endothelium is a critical step in metastasis formation.

<p>Metastatic spread of cancers occurs via a sequential process that begins with the invasion of primary tumor cells. After crossing the basement membrane and migrating through the adjacent connective tissue, certain tumor cells intravasate into tumor microvessels and circulate with the blood stream to distant organs. At the future metastatic site, these circulating tumor cells (CTC) are slowed down from the blood stream and adhere to vascular endothelium. This step is crucially initiated by interactions between endothelial selectins and certain carbohydrate ligands such as sialylated Lewis structures presented at the tumor cell surface. Adhesion is a prerequisite for extravasation and subsequent colonisation of the metastatic tissue. Selectin-binding aptamers that impair the interaction between selectin ligands and endothelial selectins would be therefore a promising new anti-metastatic therapeutic.</p

Affinities of selected DNA aptamers to rh E- and rh P-selectin determined via filter retention assays (FRA).

<p>DNA was radiolabeled, incubated with increasing amounts of proteins and filtrated through a nitrocellulose membrane. Fractions of bound DNAs were detected via autoradiography and quantified. (A) Recombinant human E-selectin incubated with DNA pool after one (▴) and 17 (•) SELEX rounds. (B) Aptamer SDA incubated with rh E-selectin (•, <i>K</i>_d ≈ 87 nM), rh P-selectin (▪, <i>K</i>_d ≈ 84 nM), or streptavidin (▴) as a control. A control DNA did neither bind to human E- (▾) nor P-selectin (♦).</p

SDA inhibited dynamic cell adhesion.

<p>Immobilized rh E- (A) or rh P- (B) selectin (0.2 μM each) as well as stimulated HPMECs (C) were incubated with 5 μM of SDA or control DNA. Selectin ligand-presenting tumor cells were perfused over immobilized proteins or E-selectin presenting HPMECs (flow rate 8 mL/h) and retaining cells were counted. SDA reduced the adhesion to corresponding cells (n = 6 of overall 2 different experiments, <i>P</i>-values applied to the selectins). IgG-Fc control showed no adhesion effect. All <i>P</i> values were calculated with untreated selectins as standard <i>(* P<0.05, ** P<0.01, *** P<0.001).</i></p

Systematic Evolution of Ligands by Exponential Enrichment (SELEX).

<p>DNA was incubated with immobilized rh E-selectin on magnetic beads (target beads). After washing and removing unbound DNA, target bound DNA was eluted and amplified via PCR. The double-stranded PCR product was separated in single strand DNA and the next SELEX step started after this regeneration. The identification of aptamers via cloning and sequencing of the enriched ssDNA pool was performed after several SELEX rounds (10–20 rounds).</p

Stability of SDA in cell culture media.

<p>Radiolabeled SDA was incubated with full medium and analyzed via polyacrylamide gel electrophoresis. After one hour more than half of SDA was still available (small box).</p

Structure and target interaction of a G-quadruplex RNA-aptamer

G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A. These RNA aptamers are specific for human interleukin-6 receptor (hIL-6R), a key player in inflammatory diseases and cancer, and have recently been exploited for in vitro drug delivery studies. With the aim to resolve the RNA structure, global shape, RNA:protein interaction site and binding stoichiometry, we now investigated AIR-3 and AIR-3A by different methods including RNA structure probing, Small Angle X-ray scattering and microscale thermophoresis. Our findings suggest a broader spectrum of folding species than assumed so far and remarkable tolerance toward different modifications. Mass spectrometry based binding site analysis, supported by molecular modeling and docking studies propose a general Gquadruplex affinity for the target molecule hIL-6R

Fluorophore Binding Aptamers as a Tool for RNA Visualization

Fluorescence correlation spectroscopy (FCS) is suitable for the detection of fluorescent molecules in living cells. For the visualization of mRNA, we genetically fused a fluorophore-specific RNA aptamer to the coding mRNA of the green fluorescent protein, as well as to noncoding sequences. Using these constructs, we showed that the aptamer portion of the mRNA still binds the fluorophore in the nanomolar range as determined via FCS. Furthermore, the binding took place in the context of total RNA extract. A tandem construct of the RNA aptamer even exhibited a lower Kd than the monomer. This FCS-based method establishes a tool for minimal invasive detection of RNA at the single molecule level in individual living cells