SELECTION OF A RNA APTAMER THAT BINDS TO HUMAN ACTIVATED PROTEIN-C AND INHIBITS ITS PROTEASE FUNCTION

A high-affinity RNA aptamer to human activated protein C (APC) was sel ected from a pool of random sequences using in vitro selection. Activa ted protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After se ven rounds of selection and amplification, a single predominant nuclei c acid sequence APC-167, a 167-base oligonucleotide with a random sequ ence core of 120 bases, was obtained. The selected aptamer did not bin d to thrombin or factor Xa and thus demonstrated specificity to APC. F urthermore, this aptamer was a non-competitive inhibitor to the cleava ge reaction of a fluorogenic substrate catalyzed by APC. The inhibitio n constant (K-i) of APC-167 was 83 nM. The 99-base oligonucleotide (AP C-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (K-i = 137 nM). Two stem-loop struc tures and at least one G . U wobble base pair in the stem were elucida ted as important structural motifs for binding.