Enzymatic polymerisation involving 2'-amino-LNA nucleotides

Area of Science:

• Molecular Biology
• Nucleic Acid Chemistry

Background:

• 2'-amino-LNA (locked nucleic acid) is a modified nucleotide with potential applications in DNA synthesis.
• Enzymatic DNA synthesis relies on polymerase activity and substrate recognition.

Purpose of the Study:

• To synthesize 2 '-amino-LNA-TTP and evaluate its utility as a substrate for DNA polymerase.
• To assess the use of 2 '-amino-LNA-T modified oligonucleotides as templates for enzymatic DNA synthesis.

Main Methods:

• Synthesis of 2 '-amino-LNA-TTP.
• Incorporation of 2 '-amino-LNA-T phosphoramidites into DNA oligonucleotides.
• Enzymatic DNA synthesis using KOD, KOD XL, and Phusion polymerases with modified substrates and templates.

Main Results:

• 2 '-amino-LNA-TTP was a suitable substrate for Phusion® HF DNA polymerase, enabling modified DNA synthesis from unmodified templates.
• Using 2 '-amino-LNA-T modified DNA as templates or 2 '-amino-LNA-TTP as a substrate decreased reaction rates and yields.
• The reduction in efficiency was more pronounced in sequences with multiple 2 '-amino-LNA-T nucleotides.

Conclusions:

• 2 '-amino-LNA-TTP can be enzymatically incorporated into DNA, but with reduced efficiency.
• Modified DNA templates containing 2 '-amino-LNA-T also hinder enzymatic synthesis of unmodified DNA.
• Further optimization is needed for efficient use of 2 '-amino-LNA-T in enzymatic DNA synthesis applications.