Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function


  • Correspondence to S. Nishikawa, National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki, Japan 305

  • Fax: +81 298 54 6095.


  • Abbreviations. APC, activated protein C; Boc, tert-butoxycarbonyl; -NH-Mec, methylcoumaryl-7-amide.

  • Enzymes. T7 RNA polymerase ( EC2.7.7.6); Taq DNA polymerase, reverse transcriptase ( EC2.7.7.7); T4 polynucleotide kinase ( EC2.7.1.78); T4 RNA ligase ( EC6.5.1.3); RNase T1 ( EC3.1.27.3); RNase A ( EC3.1.27.5).


A high-affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin-like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC-167, a 167-base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non-competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC-167 was 83 nM. The 99-base oligonucleotide (APC-99) derived from APC-167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem-loop structures and at least one G times, dot U wobble base pair in the stem were elucidated as important structural motifs for binding.