Structure/function analysis of an RNA aptamer for hepatitis C virus NS3 protease

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Hepatitis C virus (HCV) NS3 protease is a key target for antiviral therapies.
• RNA aptamers offer a potential therapeutic strategy against HCV.
• Previous studies identified RNA aptamers G9-I, -II, and -III that inhibit HCV NS3 protease.

Purpose of the Study:

• To characterize the structure-function relationship of the G9-I RNA aptamer.
• To identify the minimal functional fragment of the G9-I aptamer.
• To elucidate the tertiary interactions essential for G9-I aptamer activity.

Main Methods:

• Site-directed mutagenesis to probe structural requirements.
• In vitro selection and characterization of RNA aptamers.
• Nucleotide analog interference mapping (NAIM) to identify critical nucleotides and interactions.
• Identification of minimal inhibitory fragment (DeltaNEO-III).

Main Results:

• Mutations in stem I and stem-loop III disrupted the G9-I aptamer's active conformation and NS3 protease inhibition.
• A 51-nucleotide fragment (DeltaNEO-III) retained potent inhibitory activity, representing the minimal functional unit.
• NAIM and mutagenesis revealed an essential A-minor motif involving stem I and stem-loop III, crucial for G9-I function.

Conclusions:

• The interaction between stem I and stem-loop III is critical for the G9-I aptamer's inhibitory function against HCV NS3 protease.
• The G9-I aptamer and its minimal fragment DeltaNEO-III show promise as anti-HCV therapeutic agents.
• Understanding these structural-functional relationships aids in the rational design of aptamer-based antivirals.