Crystal structure of a closed ternary complex of a HNA Reverse Transcriptase in complex with a HNA/DNA duplex

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• 1,5-Anhydrohexitol nucleic acid (HNA) is a synthetic nucleic acid with favorable properties for applications like aptamers and catalysts.
• Efficient polymerases are needed to process HNA, particularly HNA reverse transcriptases for SELEX workflows.
• Structural insights into HNA-processing enzymes are essential for further engineering.

Purpose of the Study:

• To determine the crystal structure of an engineered HNA reverse transcriptase, KOD-H4, in a closed ternary complex.
• To understand the structural basis for HNA processing by reverse transcriptases.
• To guide future enzyme engineering efforts for improved HNA processing.

Main Methods:

• X-ray crystallography (2.8 Å resolution) of the engineered KOD-H4 reverse transcriptase.
• Formation of a closed ternary complex with dATP, a 3'-terminated primer, and a mixed HNA/DNA template.
• Comparison with a previously reported open ternary KOD-H4 structure.

Main Results:

• The crystal structure reveals KOD-H4 in a closed ternary complex, resembling wild-type enzyme pre-catalytic states.
• Increased finger and thumb domain closure and canonical base pairing at the insertion site were observed.
• HNA residue conformation changed from 1C4 to 4C1, indicating state-dependent conformational flexibility of HNA.

Conclusions:

• Engineered KOD-H4 can form a closed, pre-catalytic complex similar to wild-type enzymes with natural substrates.
• The study reveals the conformational flexibility of HNA during reverse transcription.
• Understanding HNA flexibility is critical for designing and optimizing enzymes that process HNA.