Optimization of an unnatural base pair toward natural-like replication

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Efforts to expand the genetic alphabet focus on unnatural nucleotides for DNA replication.
• A previously identified unnatural base pair (dMMO2:d5SICS) showed promise but required optimization for efficiency and fidelity.

Purpose of the Study:

• To optimize the replication of the dMMO2:d5SICS unnatural base pair.
• To synthesize and evaluate dMMO2 derivatives (d5FM and dNaM) for improved polymerase-mediated replication.

Main Methods:

• Synthesis of two dMMO2 derivatives, d5FM and dNaM.
• Evaluation of the insertion efficiency and fidelity of these derivatives opposite d5SICS using DNA polymerase.
• Comparison of replication efficiency and fidelity with the parent dMMO2:d5SICS pair and natural base pairs.

Main Results:

• Both d5FM and dNaM derivatives showed higher insertion efficiency opposite d5SICS compared to dMMO2.
• The resulting unnatural base pairs (d5FM:d5SICS and dNaM:d5SICS) exhibited improved replication efficiency and fidelity.
• The dNaM:d5SICS heteropair demonstrated replication efficiency approaching natural base pairs, with fidelities ranging from 10^3 to 10^4.

Conclusions:

• Modified unnatural nucleotides (d5FM, dNaM) significantly enhance the replication of unnatural base pairs.
• The dNaM:d5SICS pair represents a substantial advancement towards natural-like replication efficiency and fidelity.
• A generalized model for unnatural base pair replication was proposed, aiding future design of synthetic genetic elements.