Exploring TAR-RNA aptamer loop-loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon...

Area of Science:

• Structural Biology
• Virology
• RNA Biology

Background:

• The human immunodeficiency virus type 1 (HIV-1) trans-activation responsive (TAR) RNA element regulates viral gene transcription.
• TAR RNA is a critical binding site for viral and cellular proteins, making it a target for antiviral strategies.
• Previous studies selected RNA aptamers forming kissing-loop dimers with TAR to interfere with the viral life cycle.

Purpose of the Study:

• To elucidate the three-dimensional structure of the HIV-1 TAR RNA element in complex with a high-affinity RNA aptamer.
• To characterize the binding interface and conformational details of the TAR-aptamer kissing complex.
• To investigate the role of specific intermolecular interactions in stabilizing the complex.

Main Methods:

• X-ray crystallography was employed to determine the high-resolution structure of the TAR-aptamer complex.
• Thermal denaturation (Tm) assays were used to assess complex stability.
• Surface plasmon resonance (SPR) was utilized to quantify binding affinities and kinetics.
• Chemical modifications (2'-O-methyl) were introduced to probe the role of hydrogen bonds.

Main Results:

• The crystal structure revealed a kissing-loop dimer formed between TAR RNA and the aptamer, interacting via six Watson-Crick base pairs.
• The complex adopted a unique conformation with an inter-helix angle of 28.1 degrees, differing from previous solution and modeling studies.
• Structural analysis identified potential inter-backbone hydrogen bonds involving ribose 2' hydroxyl and phosphate groups at the stem-loop junctions.
• Experimental data confirmed the involvement of these intermolecular hydrogen bonds in stabilizing the TAR-aptamer complex.

Conclusions:

• The crystal structure provides unprecedented atomic detail of the HIV-1 TAR RNA-aptamer complex.
• The observed conformation and stabilizing hydrogen bonds offer insights into RNA-RNA recognition mechanisms relevant to viral regulation.
• Understanding these interactions can inform the design of novel aptamer-based therapeutics targeting HIV-1 replication.