Enzymatic polymerization of phosphonate nucleosides.

Area of Science:

• Molecular Biology
• Biochemistry
• Nucleic Acid Chemistry

Background:

• The Therminator polymerase efficiently processes 5'-O-phosphonomethyl-2'-deoxyadenosine (PMdA).
• The substrate capabilities of phosphonate nucleoside analogues (PMdN) with natural DNA polymerases remain largely unexplored.

Purpose of the Study:

• To investigate the substrate potential of PMdA, PMdC, PMdT, and PMdU analogues for natural DNA polymerases.
• To evaluate the enzymatic incorporation and chain elongation capabilities of these modified nucleosides.

Main Methods:

• Enzymatic polymerization assays using various natural DNA polymerases.
• Kinetic analysis of PMdA incorporation by Vent(exo(-)) polymerase.
• Synthesis of DNA duplexes containing modified nucleosides.

Main Results:

• PMdA and PMdC were incorporated with complete chain elongation by all tested DNA polymerases.
• PMdT and PMdU showed limited enzymatic polymerization.
• Mixed DNA sequences with modified A, C, and T residues were synthesized using Vent(exo(-)) and Therminator polymerases.
• PMdA exhibited a reduced K(M) value for incorporation by Vent(exo(-)) polymerase compared to natural substrates.

Conclusions:

• Certain phosphonate nucleoside analogues, specifically PMdA and PMdC, are efficiently processed by natural DNA polymerases.
• These findings support the potential for directed evolution of polymerases to enhance the synthesis of modified DNA strands.
• The study highlights the utility of PMdA and PMdC as building blocks for novel DNA structures.