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Journal of Virology, April 2002, p. 3688-3696, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3688-3696.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Selection of RNA Aptamers That Are Specific and High-Affinity Ligands of the Hepatitis C Virus RNA-Dependent RNA Polymerase

Antonino Biroccio,1 Jörg Hamm,2,{dagger} Ilario Incitti,1 Raffaele De Francesco,1 and Licia Tomei1*

Istituto di Ricerche di Biologia Molecolare "P. Angeletti," 00040 Pomezia-Rome, Italy,1 Department of Biochemistry, University of Dundee, Dundee DD1 5EH, Scotland2

Received 20 September 2001/ Accepted 2 January 2002

In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5B{Delta}C55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.

* Corresponding author. Mailing address: Istituto di Ricerche di Biologia Molecolare "P. Angeletti" (IRBM), Via Pontina Km 30,600, 00040 Pomezia-Rome, Italy. Phone: 39-06-91093230. Fax: 39-06-91093225. E-mail: Licia_Tomei{at}Merck.com.

{dagger} Present address: Dipartimento di Genetica, Biologia e Biochimica, Università degli Studi di Torino, 10126 Turin, Italy.

Journal of Virology, April 2002, p. 3688-3696, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3688-3696.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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