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Journal of Biochemistry Advance Access published online on October 8, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj203
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© 2006 The Japanese Biochemical Society.
Received August 10, 2006
Accepted September 18, 2006

Regular Paper

An RNA Aptamer That Discriminates Bovine Factor IX from Human Factor IX

Subash C. B. Gopinath 1, Dhakshnamoorthy Balasundaresan 1, Joe Akitomi 2, and Hiroshi Mizuno 2 *

1 Functional Nucleic Acids Group, Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba City 305-8566, Ibaraki, Japan
2 VALWAY Technology Center, NEC Soft Co. Ltd., Shinkiba 1-18-7, Tokyo 136-8627, Japan

* To whom correspondence should be addressed.
Hiroshi Mizuno, E-mail: mizuno-hiroshi{at}aist.go.jp


  Abstract

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to106. The discriminating ability of this mini aptamer is calculated as more than 1000 fold. The equilibrium dissociation constant (Kd) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.

Keywords: aptamer; bovine factor IX; discrimination; human factor IX; RNA; SELEX.
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