Selective and stable base pairing by alkynylated nucleosides featuring a spatially-separated recognition interface

Area of Science:

• Synthetic biology
• Biochemistry
• Molecular biology

Background:

• Unnatural base pairs (UBPs) are crucial for advancing nucleic acid-based technologies due to their selective pairing.
• Conventional UBP designs often rely on modified Watson-Crick interfaces.
• A new approach is needed to expand the utility of UBPs.

Purpose of the Study:

• To demonstrate exclusive base pairing through spatial separation of recognition units, an alternative to conventional UBP designs.
• To develop and characterize novel alkynylated purine and pyridazine nucleosides with pseudo-nucleobases.
• To assess the stability and sequence-specificity of these novel UBPs in DNA duplexes.

Main Methods:

• Design and synthesis of alkynylated purine (NPu, OPu) and pyridazine (NPz, OPz) nucleosides.
• Incorporation of pseudo-nucleobases (2-aminopyrimidine or 2-pyridone) on the major groove side.
• Characterization using melting temperature (Tm) measurements, 2D-NMR, and molecular dynamics (MD) simulations.

Main Results:

• Alkynylated purine-pyridazine pairs formed stable, exclusive hydrogen bonds via pseudo-nucleobases in the DNA major groove.
• Consecutive incorporation of these UBPs significantly stabilized DNA duplexes.
• High sequence-specificity was maintained throughout the stabilization process.

Conclusions:

• Spatial separation of recognition units is a viable strategy for designing novel UBPs.
• The developed alkynylated purine-pyridazine pairs offer enhanced stability and specificity for nucleic acid technologies.
• This approach broadens the scope for creating custom DNA functionalities.