Structural and mechanistic insights into modified G-quadruplex thrombin-binding DNA aptamers

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• Thrombin-binding aptamers (TBAs) are crucial for thrombin interaction, folding into G-quadruplex structures.
• Modifications at position 7 of the TGT loop, specifically with unlocked uracil (UNA), d-isothymidine (D-isoT), or l-isothymidine (L-isoT), alter TBA activity.
• The precise mechanisms by which these modifications influence TBA biological properties remain largely unexplored.

Purpose of the Study:

• To elucidate the structural variations and binding modes of modified TBAs using D-isoT, L-isoT, and UNA.
• To understand how these modifications impact the structure-activity relationships of thrombin-binding aptamers.
• To provide insights for the rational design of novel modified aptamers.

Main Methods:

• Molecular dynamics (MD) simulations were employed to investigate structural changes and binding interactions.
• Free energy calculations were performed to quantify interaction strengths.
• Comparative structural analyses focused on the TGT loop conformation and protein interactions.

Main Results:

• D-isoT and UNA modifications altered the TGT loop conformation, leading to stronger interactions with thrombin.
• D-isoT and UNA exhibited similar conformations, correlating with their comparable biological activities.
• L-isoT modification induced a tendency for the aptamer to unfold.
• Analysis revealed distinct flexibility patterns in the TT loops for each modification.

Conclusions:

• D-isoT and UNA modifications enhance TBA binding affinity through specific conformational changes.
• L-isoT destabilizes the aptamer structure, negatively impacting its function.
• These findings facilitate a structure-based approach for designing improved aptamers.