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Nucleic Acids Research Advance Access published online on January 9, 2009
Nucleic Acids Research, doi:10.1093/nar/gkn1012
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A designed RNA selection: establishment of a stable complex between a target and selectant RNA via two coordinated interactions

Tomoaki Shiohara1, Hirohide Saito1,2,* and Tan Inoue1,2,*

1Laboratory of Gene Biodynamics, Graduate School of Biostudies, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502 and 2ICORP, Japan Science and Technology Agency (JST), 5 Sanban-cho, Chiyoda-ku, Tokyo 102-0075, Japan

*To whom correspondence should be addressed. Tel: +81 75 753 3995; Fax: +81 75 753 3996; Email: h-saito{at}kuchem.kyoto-u.ac.jp

Correspondence may also be addressed to Tan Inoue. Email: tan{at}kuchem.kyoto-u.ac.jp

Received November 6, 2008. Accepted December 4, 2008.

In this paper, we describe a new method for selecting RNA aptamers that cooperatively bind to two specific sites within a target RNA. We designed a selection system in which two RNAs, a target RNA and a RNA pool, were assembled by employing a pre-organized GAAA tetraloop-11-nt receptor interaction. This allows us to select the binding sequence against a targeted internal loop as well as a linker region optimized for binding of the two binding sites. After the selection, the aptamers bound with dissociation constants in the nanomolar range, thereby forming a stable complex with the target RNA. Thus this method enables identification of aptamers for a specific binding site together with a linker for cooperative binding of the two RNAs. It appears that our new method can be applied generally to select RNAs that adhere tightly to a target RNA via two specific sites. The method can also be applicable for further engineering of both natural and artificial RNAs.


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