Sanger Sequencing of Xeno-Nucleic Acid

Area of Science:

• Biotechnology
• Synthetic Biology
• Molecular Biology

Background:

• Xeno-nucleic acids (XNAs) are synthetic genetic polymers with enhanced stability compared to DNA.
• Current DNA sequencing technologies are incompatible with XNAs, limiting their application.
• Developing methods for XNA sequencing is crucial for their use in biotechnology and information technology.

Purpose of the Study:

• To develop a direct Sanger sequencing method for XNA.
• To enable accurate sequence determination of various XNA types.
• To lay the groundwork for XNA-based applications like data storage.

Main Methods:

• Utilized a Bst DNA polymerase F710Y mutant engineered for XNA template recognition.
• Employed a chain termination strategy with dideoxyribonucleoside triphosphates (ddNTPs) for complementary DNA synthesis.
• Demonstrated proof-of-concept automated sequencing on a genetic analyzer using BigDye-labeled ddNTPs.

Main Results:

• Achieved direct readout of XNA strands up to 50 bases in length.
• The Bst F710Y mutant showed significantly enhanced activity in ddNTP incorporation.
• Accurate sequencing was demonstrated for XNAs with different backbone chemistries (e.g., TNA, FANA).

Conclusions:

• Established a novel Sanger sequencing platform for XNA analysis.
• The method facilitates de novo identification and characterization of functional XNAs.
• Paves the way for advanced XNA applications, including genetic information storage.