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 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 x U wobble base pair in the stem were elucidated as important structural motifs for binding.
Gal
S W
SW
National Institute of Bioscience and Human Technology, AIST, MITI, Tsukuba Science City, Ibaraki, Japan.
Amontov
S
S
Urvil
P T
PT
Vishnuvardhan
D
D
Nishikawa
F
F
Kumar
P K
PK
Nishikawa
S
S
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D005038:Ethylnitrosourea; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011486:Protein C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D009843:Oligoribonucleotides; D011486:Protein C; D012313:RNA; D005038:Ethylnitrosourea