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Nucleic Acids Research, 2003, Vol. 31, No. 7 1935-1943
© 2003 Oxford University Press

Inhibition of HCV NS3 protease by RNA aptamers in cells

Fumiko Nishikawa, Nobuko Kakiuchi, Kohei Funaji1, Kotaro Fukuda, Satoru Sekiya and Satoshi Nishikawa

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Sciences and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan and 1 Mitsubishi Gas Chemical Company, Inc., Marunouchi, Tokyo 100-8324, Japan

Nobuko Kakiuchi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan

Non-structural protein 3 (NS3) of hepatitis C virus (HCV) has two distinct activities, protease and helicase, which are essential for HCV proliferation. In previous work, we obtained RNA aptamers (G9-I, II and III) which specifically bound the NS3 protease domain ({Delta}NS3), efficiently inhibiting protease activity in vitro. To utilize these aptamers in vivo, we constructed a G9 aptamer expression system in cultured cells, using the cytomegarovirus enhancer + chicken ß-actin globin (CAG) promoter. By conjugating the cis-acting genomic human hepatitis delta virus (HDV) ribozyme and G9-II aptamer, a chimeric HDV ribozyme-G9-II aptamer (HA) was constructed, which was used to produce stable RNA in vivo and to create tandem repeats of the functional unit. To target the transcribed RNA aptamers to the cytoplasm, the minimal mutant of constitutive transport element (CTE), derived from type D retroviruses, was conjugated at the 3' end of HA (HAC). Transcript RNAs from (HA)n and (HAC)n were processed into the G9-II aptamer unit by the cis-acting HDV ribozyme, both in vitro and in vivo. Efficient protease inhibition activity of HDV ribozyme-G9-II aptamer expression plasmid was demonstrated in HeLa cells. Protease inhibition activity level of tandem chimeric aptamers, (HA)n and (HAC)n, rose with the increase of n from 1 to 4.


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