A new way of targeting protease-receptor binding: Selection of uPA binding RNA aptamers inhibiting its association to its receptor uPAR
Abstract:The binding of urokinase-type plasminogen activator (uPA) to its cell surface receptor (uPAR) has been implicated in tumour spread. We have employed a new method for developing inhibitors of the binding. Systematic evolution of ligands by exponential enrichment (SELEX) is an approach to selecting for RNA or DNA oligonucleotides with specific properties. Selections can be performed from a large pool of random sequences (1015) of RNA or DNA oligonucleotides which fold into three-dimensional structures. It is then possible to select RNA or DNA oligonucliotides binding with high affinities to targets of interest. These binding specific oligonucleotides are called aptamers. In a SELEX experiment, we used a library of serum-stable 2'-fluoro-pyrimidine modified RNA oligonucleotides to select for RNA aptamers capable of binding to urokinase-type plasminogen activator (uPA). Analysed by surface plasmon resonance (SPR), the selected aptamers bind to the amino terminal fragment of human uPA with KD-values in the low nanomolar range. Cell binding assays showed that the selected aptamers were able to inhibit the binding of the amino terminal fragment of uPA to the receptor uPAR expressed on U937 cells. The same conclusion was reached by SPR analyse. The aptamers showed no inhibition of uPA's proteolytic activity. Based on computerised secondary structure predictions, it was possible to reduce size of the aptamers down to 49 nucleotides without losing the inhibitory properties. uPA-binding aptamers could be a promising tool for interfering with the pathophysiological functions of the plasminogen activation system. The inhibition of the uPA-uPAR interaction will inhibit plasminogen activation on the cells surface without interfering with the other function of uPAR and uPA. Aptamers are also interesting tools for analytic and imaging purpose because of the small size and high affinities.
Document Type: Abstract
Affiliations: Department of Molecular Biology
Publication date: May 1, 2008