Unusual Base Pair between Two 2-Thiouridines and Its Implication for Nonenzymatic RNA Copying

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Biology

Background:

• 2-Thiouridine (s²U) is a modified nucleobase known to improve RNA efficiency and accuracy.
• Understanding RNA base pairing is crucial for RNA replication and function.

Purpose of the Study:

• To investigate the structural and thermodynamic properties of the unusual 2-thiouridine:2-thiouridine (s²U:s²U) base pair.
• To assess the impact of the s²U:s²U base pair on nonenzymatic RNA copying fidelity.

Main Methods:

• High-resolution crystal structure analysis of RNA duplexes containing s²U:s²U pairs.
• Thermodynamic stability measurements of s²U:s²U versus U:U base pairs.
• Competition experiments to evaluate misincorporation rates during nonenzymatic RNA template copying.

Main Results:

• A novel, stable s²U:s²U base pair was identified, comparable in stability to native A:U pairs.
• The s²U:s²U pair features a C═S···H-N hydrogen bond, distinct from the U:U pair's C═O···H-N bond.
• Despite its stability, s²U:s²U self-pairing minimally increased misincorporation due to competition from correct A:s²U pairing and subsequent base addition stalling.

Conclusions:

• The s²U:s²U base pair enhances RNA duplex stability and RNA copying fidelity.
• s²U modification improves nonenzymatic RNA copying rates and efficiency with minimal fidelity cost.
• This modification offers a promising strategy for improving RNA replication fidelity in prebiotic or synthetic systems.