Optimization of unnatural base pair packing for polymerase recognition

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Efforts to expand the genetic alphabet are ongoing.
• Previously, the synthesis and thermal stability of unnatural base pairs using methyl-substituted phenyl ring nucleobase analogues were reported.
• Some of these pairs demonstrated stability and selectivity comparable to natural base pairs.

Purpose of the Study:

• To characterize the polymerase-mediated replication of unnatural base pairs formed by hydrophobic nucleobase analogues.
• To investigate the role of hydrogen bonding and aromatic surface area in polymerase recognition.
• To assess the potential of phenyl nucleobase analogues for creating a third base pair.

Main Methods:

• Synthesis of unnatural base pairs with methyl-substituted phenyl ring nucleobase analogues.
• Thermal stability analysis of these base pairs in duplex DNA.
• Characterization of polymerase-mediated replication, including correct and incorrect base pair synthesis and primer extension.

Main Results:

• Polymerase-mediated replication is sensitive to the specific methyl substitution pattern of the nucleobase analogue.
• Neither hydrogen bonding nor large aromatic surface area is essential for polymerase recognition.
• Interstrand interactions between small aromatic rings can be optimized for replication.

Conclusions:

• Appropriately derivatized phenyl nucleobase analogues show promise for developing a third base pair.
• These analogues offer a viable strategy for expanding the genetic alphabet.
• The findings advance the understanding of DNA replication mechanisms with unnatural base pairs.