A strongly pairing fifth base: oligonucleotides with a C-nucleoside replacing thymidine

Area of Science:

• Synthetic biology
• Nucleoside chemistry
• Molecular biology

Background:

• DNA and RNA contain five canonical bases.
• Thymine and uracil form two hydrogen bonds with adenine.
• A:T rich duplexes are less stable than C:G rich duplexes, complicating hybridization-based detection.

Purpose of the Study:

• To synthesize a novel nucleoside analog with enhanced base-pairing stability.
• To create a thymidine replacement that pairs as strongly as G:C pairs.
• To develop a tool for targeting A-rich sequences in biological studies.

Main Methods:

• Synthesis of an ethynylmethylpyridone C-nucleoside (W) building block.
• Incorporation of W residues into oligonucleotides via automated synthesis.
• Melting point analysis of DNA duplexes containing W residues.

Main Results:

• The synthesized W nucleoside pairs with adenine as strongly as cytosine pairs with guanine.
• Oligonucleotides containing W showed increased melting points by up to 17.5°C compared to T:A pairs.
• The W base demonstrated high fidelity, with a single G-W mismatch causing a significant melting point decrease.

Conclusions:

• The W nucleoside is a stable, high-fidelity replacement for thymidine.
• This synthetic base enhances the stability of DNA duplexes.
• W can be utilized to target and study A-rich sequences in genomic applications.