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

• Published in: European journal of biochemistry Vol. 252; no. 3; pp. 553 - 562
• Main Authors: Gal, Sang Wan, Amontov, Sergey, Urvil, Petri Tapani, Vishnuvardhan, Daesety, Nishikawa, Fumiko, Kumar, Penmetcha K. R., Nishikawa, Satoshi
• Format: Journal Article
• Language: English
• Published: Berlin & Heidelberg Springer‐Verlag 15.03.1998
• Subjects: activated protein C; aptamer; aptamers; Base Composition; Base Sequence; Binding Sites; C protein; coagulation factor Xa; DNA Primers; Ethylnitrosourea; Humans; in vitro selection; Kinetics; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligoribonucleotides - chemistry; Oligoribonucleotides - metabolism; Polymerase Chain Reaction; Protein C - antagonists & inhibitors; Protein C - metabolism; RNA; RNA - chemistry; RNA - metabolism; serine proteinase; thrombin; trypsin; trypsin‐like serine protease
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1327.1998.2520553.x

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 U wobble base pair in the stem were elucidated as important structural motifs for binding.