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

Abstract

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 29-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 29-fluoro-pyrimidine-modified RNA aptamers represent a novel promising principle for interfering with the pathological functions of the uPA system.

Targeting Tumor Cell Invasion and Dissemination In Vivo by an Aptamer That Inhibits Urokinase-type Plasminogen Activator through a Novel Multifunctional Mechanism

“…By using full-length human uPA as a target, we previously selected RNA aptamers, which were all directed against the ATF of uPA [37]. Similar to upanap-126, several of the ATF-binding aptamers were found to inhibit the uPA-uPAR interaction [37].…”

“…The RNA library was constructed with a 35-nucleotide degenerate sequence and with 2′-fluor-pyrimidines as described [37] using the primers 5′-CGCGGATCCTAATACGACTCACTATAGGGGCCACCAACGACATT-3′ containing the T7 promoter sequence and 5′-GATCCATGGGCACTATTTATATCAAC(N 35 )AATGTCGTTGGTGGCCC-3′ containing the degenerate sequence. Enrichment of RNA aptamers with binding affinity towards the truncated version of human pro-uPA (residues 134–411), lacking the EGF and kringle domains and harbouring the mutation S356A, was carried out by capturing the protein target on protein A-coupled Sepharose beads using the rabbit polyclonal anti-uPA antibody F1609 [34] essentially as described [37] with 12 rounds of enrichments. To obtain 2′-fluoro-pyrimidine modified RNA molecules, RNA transcriptions were carried out using 2.5 mM each of ATP and GTP (GE Healthcare), and 2.5 mM each of 2′-F-dCTP and 2′-F-dUTP (2′-F-Y, TriLink Biotechnologies) and 50 μg/mL mutant T7 RNA polymerase Y639F.…”

Nucleic Acid Aptamers Against Proteases

“…Dupont et al (72) screened a library of 2´-Fpyrimidine modified RNA aptamers with human uPA and isolated several aptamers which were able to block the binding of uPA to uPAR with IC 50 values in the low nanomolar range, but none of the aptamers affected the ability of uPA to catalyze the conversion of plasminogen to plasmin. The aptamers did not cross-react with mouse uPA.…”

“…The degree of truncation possible without loss of affinity and/or inhibitory activity however appears highly variable. For example, the APC aptamer APC-167 could only be reduced from 167 to 99 nucleotides (65), the FVII aptamer 16.3 from 80 to 56 nucleotides (56), and the uPA aptamer from 79 to 49 nucleotides (72). In a few cases, reduction in size was even accompanied by an increased affinity as exemplified by the thrombinbinding RNA aptamers Toggle25, which could be reduced from 96 to 25 nucleotides with a change in K D from 3 to 0.5 nM for human thrombin, and RNA 16.24, which could be reduced from 79 to 24 nucleotides with a change in K D from 37 to 9 nM, However, even in the absence of an affinity loss, size reduction may modulate the functionality and binding specificity.…”

“…They can also have speciesspecificity as found for bovine FIX aptamers preventing bovine but not measurably human thrombin-catalyzed fibrinogen conversion (58). Likewise, uPA-uPAR blocking uPA aptamers were able to inhibit the interaction in a human but not a mouse setting (72). For therapeutic applications species crossreactivity is desired to enable animal testing.…”