Evoking picomolar binding in RNA by a single phosphorodithioate linkage

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RNA aptamers are synthetic affinity molecules with potential therapeutic and diagnostic applications.
• Current RNA aptamers often lack sufficient binding affinity and specificity for practical use.
• Enhanced binding affinity is crucial for improving downstream aptamer applications.

Purpose of the Study:

• To investigate the use of phosphorodithioate (PS2) substitution in RNA aptamers to enhance target binding affinity.
• To elucidate the structural basis for the improved binding affinity.

Main Methods:

• Chemical modification of RNA aptamers with phosphorodithioate (PS2) substitution.
• X-ray co-crystallography of the modified aptamer complexed with its target (α-thrombin).
• High-level quantum mechanical calculations on model systems.

Main Results:

• PS2 substitution dramatically improved RNA aptamer binding affinity by approximately 1000-fold (nanomolar to picomolar).
• X-ray crystallography revealed a localized induced-fit rearrangement at the PS2-modified nucleotide enhancing target interaction.
• Quantum mechanical calculations indicated favorable sulfur-aromatic ring interactions and increased polarizability of sulfur analogs.

Conclusions:

• Phosphorodithioate substitution is a powerful strategy to significantly enhance RNA aptamer binding affinity.
• The structural and electronic properties of the sulfur atom in PS2 are key to the improved aptamer-target interactions.
• This modification holds promise for advancing aptamer-based diagnostics and therapeutics.