Analysis of interaction between RNA aptamer and protein using nucleotide analogs

Area of Science:

• Biochemistry
• Virology
• Molecular Biology

Background:

• Hepatitis C virus (HCV) non-structural protein 3 (NS3) is vital for viral replication.
• NS3 possesses trypsin-like serine protease and helicase functional domains.
• Targeting NS3 protease activity is a key strategy for HCV inhibition.

Purpose of the Study:

• To isolate and characterize RNA aptamers that inhibit HCV NS3 protease activity.
• To elucidate the structural features of these aptamers essential for their function.
• To identify critical nucleotides within the aptamers responsible for binding and inhibition.

Main Methods:

• In vitro selection (SELEX) was employed to obtain RNA aptamers.
• Secondary structure modeling (Mulfold) was used to predict aptamer structures.
• Modification interference analysis with nucleotide analogs was performed.

Main Results:

• Three RNA aptamers (G9-I, -II, -III) targeting the NS3 protease domain (delta NS3) were isolated.
• These aptamers demonstrated strong inhibition of NS3 protease activity.
• A common sequence (5'-GA(A/U)UGGGAC-3') and distinct junction structures (three-way and four-way) were identified.
• Modification interference analysis pinpointed common essential nucleotides across all three aptamers.

Conclusions:

• The identified RNA aptamers are potent inhibitors of HCV NS3 protease.
• Specific nucleotides and structural elements are critical for aptamer activity.
• These findings provide insights into aptamer-protein interactions for antiviral drug development.